Effect of dry ice addition and wilting on the rates of fermentation and proteolysis in ensiled alfalfa

Abstract

The aim of this study was to determine the effect of dry ice addition at different levels and herbage wilting on the rate of fermentation and changes in crude protein (CP) composition in ensiled alfalfa. Alfalfa was ensiled with the addition of the following quantities of dry ice at each degree of wilting (0-h and 12-h wilting): 0 g, 0.5 g, 1 g, 2 g. Silages were made in mini silos, which were opened after 1, 3, 7, 15, 30 days. The addition of dry ice and wilting had a significant effect on the analyzed parameters (p ≤ 0.05). The slowest decrease in true protein content was noted in silage produced after a 12-h wilting with the addition of 2 g of dry ice (p = 0.014). On fermentation day 30, silage produced after a 12-h wilting with the addition of 2 g of dry ice was characterized by the significantly lowest proportions of fractions of ammonia nitrogen (PA1) (p = 0.027), buffer-soluble true protein (PA2) (p = 0.028), and indigestible protein (PC) (p = 0.044), and the highest proportions of fractions of buffer-insoluble true protein (PB1) (p = 0.045) and fiber-bound protein (PB2) (p ≤ 0.001). The study demonstrated that the addition of 2 g of dry ice combined with a 12-h wilting prior to ensiling positively influenced the fermentation pattern and reduced proteolysis, thus improving the nutritional value of CP in alfalfa silage.

Introduction

Alfalfa (Medicago sativa L.) is one of the key components of dairy cattle diets due to its high crude protein (CP) content. Ensiling is the most popular alfalfa preservation technique. The ensiling process is complex and its effectiveness is determined by plant material characteristics including values of dry matter (DM), water-soluble carbohydrates (WSC) and buffering capacity, and the composition and population of epiphytic microbiota. Therefore, the composition of alfalfa herbage at harvest has a significant effect on its ensilability and silage quality^1,2.

During ensilage, the first phase (aerobic) of fermentation usually lasts several hours. During that time, atmospheric oxygen present between plant particles undergoes reduction due to the respiration of the plant material and aerobic and facultative aerobic micro-organisms (yeasts, enterobacteria). Plant enzymes such as proteases and carbohydrases are also active during this phase if the pH remains within the normal range for fresh forage juice (pH 6.5–6.0). The second (anaerobic) phase of fermentation lasts from several days to several weeks. Lactic acid bacteria develop and become the predominant bacterial population in this phase. The pH decreases to 3.8–5.0 due to the production of lactic acid (LA) and other acids. During the third (stable) phase of fermentation, the abundance of most microorganisms decreases steadily, and only selected acid-tolerant microbial groups survive in inactive state, whereas other microorganisms, such as Clostridia spp., survive as spores. Only some acid-tolerant proteases and carbohydrases and specialized bacteria such as Lactobacillus buchneri remain active, but the level of their activity is low³.

The CP from alfalfa is rapidly degraded in the rumen due to extensive proteolysis⁴. Some true protein (TP) is degraded to simpler compounds characterized by higher solubility such as non-protein nitrogen (NPN). Due to proteolytic activity, the amount of NPN increases from 16.7 to 19.9% CP in unwilted alfalfa to 59.1–82.6% CP or even 87.0% CP in silage⁵. Many forages experience proteolytic losses during ensilage, but they are much smaller than those recorded in alfalfa. For instance, in red clover silage, NPN can account for 7% to a maximum of 40.8% CP⁶.

During the LA fermentation of alfalfa silage, proteolytic activity is largely attributed to plant enzymes, whereas amino acid catabolism observed in later stages is attributed to microbial activity⁷. In addition, TP availability also decreases in alfalfa silage due to the increasing amount of fiber-bound protein⁸. Despite the high digestibility and palatability of alfalfa silage, increased concentrations of ammonia nitrogen (NH₃–N) in the rumen and urea in cow’s milk can be observed, pointing to poorer dietary CP utilization⁹.

The effect of wilting on the fermentation pattern and protein protection in alfalfa during ensilage has been documented in the literature^10,11. According to many researchers, the rate of biomass acidification, which can be modified by shortening the aerobic phase of fermentation, is the most important factor responsible for the extent of proteolysis⁵. The aerobic phase of fermentation can be shortened with the use of inhibitors such as organic acids or stimulants – additives with high WSC content¹². Another less researched method is the addition of dry ice as a source of carbon dioxide (CO₂). This gas is expected to rapidly remove oxygen present in ensiled alfalfa or compensate for the inadequate filling of empty spaces between particles of the ensiled forage mass with air, thus shortening the aerobic phase of fermentation. Such a mechanism can reduce the activity of aerobic microorganisms within a short period of time, minimize WSC losses, and decrease the temperature of the ensiled forage mass, thus affecting the activity of proteolytic plant enzymes¹³. For practical reasons, gaseous CO₂ cannot be used in silage production. One of the solutions is to use dry ice as a source of CO₂. Dry ice is a by-product of the chemical industry, and its largest quantities are generated during the production of NH₃–N¹⁴.

The first study investigating the use of dry ice in silage production was performed on alfalfa. The proximate chemical composition and selected fermentation parameters of alfalfa silage in response to dry ice added at 2 g to 14 g dm⁻³ were analyzed on a laboratory scale¹⁵. Nussbaum¹⁶ found that dry ice at 1 kg m⁻³ did not improve the quality of maize fermentation. In contrast, Thaysen¹⁷ reported lower nutrient losses and a more desirable fermentation pattern when maize was ensiled with the addition of dry ice. None of the few studies conducted to date have evaluated the effect of dry ice on proteolytic processes.

The research hypothesis states that CO₂ formed during dry ice sublimation, due to its higher density, removes oxygen from the voids in the ensiled alfalfa mass, shortens the aerobic phase of fermentation, and accelerates acidification, thereby reducing proteolysis.

The aim of this study was to determine the effect of dry ice addition at different levels on the rate of fermentation and changes in CP composition during ensiling of fresh and wilted alfalfa.

Materials and methods

Forage and treatments

The present experiment was performed on alfalfa silage. The plant material was obtained from a commercial farm (54°12′03″N, 20°49′33″E, Poland) and the Authors had permission to collect it. Herbicides or fertilizers were not applied. First-cut herbage was harvested mechanically using a Krone AM 283 S disc mower (Krone GmbH, Spelle, Germany), in the early bud stage, at a height of 5 cm, between noon and 1 p.m. Part of the harvested forage was collected immediately after cutting (0-h wilting), and the remaining forage was left in the field, in swaths of the same thickness, under a roofed shelter, and allowed to wilt for 12 h. The collected forage was mechanically chopped to a theoretical chaff length of 25 mm. Alfalfa was ensiled with the addition of the following quantities of dry ice at each degree of wilting (0-h wilting and 12-h wilting): 0 g, 0.5 g, 1 g, and 2 g. Alfalfa silages were made in mini silos with a volume of 1 dm³, in three replicates for each experimental treatment. During silage preparation, when 50% of the forage mass had been compacted in the silo, dry ice pellets with a diameter of 16 mm were added, after which the remaining forage was added and compacted to a final density of 750 kg fresh matter·m⁻³. The silos were equipped with exhaust pipes. Three mini silos in each experimental treatment were opened after 1, 3, 7, 15, and 30 days. All mini silos were stored in the same conditions, that is temperature 18–20 °C, humidity 55–60%.

Chemical composition analysis

Representative herbage samples were collected immediately after cutting and after 12 h of wilting. A representative silage sample was collected from each mini silo after opening. Part of the sample was frozen at − 25 °C, and the remaining part was dried at 60 °C for 48 h in a Binder FED 115 dryer (Binder, GmbH, Tuttlingen, Germany) and ground in a mill for fibrous materials (ZM 200, Retsch, Haan, Germany) to 1 mm particle size.

The content of DM and CP was determined in herbage and silage samples according to AOAC methods¹⁸. The pH of silage was measured with the HI 8314 pH-meter (Hanna Instruments, Woonsocket, Rhode Island, USA) and the concentrations of LA and short-chain fatty acids (SCFAs), including acetic acid (AA), propionic acid, isobutyric acid, butyric acid (BA), isovaleric acid, and valeric acid, and ethanol were determined as described by Kostulak-Zielińska and Potkański¹⁹, and Gąsior²⁰. For the above chemical analyses, silage samples were homogenized (1:5 ratio of sample weight per water volume, w/v), and were filtered through polyamide gauze. Next, the filtrate was again passed through a soft filter, deproteinized with a 24% solution of metaphosphoric acid, and centrifuged (13 000 rpm, 7 min). The LA content was determined by high-performance liquid chromatography (HPLC, SHIMADZU, Kyoto, Japan) with isocratic flow. Separation was carried out using the Varian METACARB 67H column (ORGANIC ACIDS COLUMN) (Varian Inc., Palo Alto, California, USA); mobile phase: 0.002 M solution of sulfuric acid in deionized water, flow rate of 1 cm³ min⁻¹, UV detector, 210 nm. External fatty acid and ethanol standards were supplied by SUPELCO (Sigma-Adrich Co. Ltd., Saint Louis, MI, USA), and the LA standard – by FLUKA (Chemie GmbH, Buchs, Switzerland). Short-chain fatty acids were separated and determined by gas chromatography (GC) on the Varian 450-GC with the Varian CP-8410 autosampler (Varian Inc., Palo Alto, California, USA), using a flame-ionization detector (FID) and a CP-FFAP capillary column (length – 25 m, inner diameter – 0.53 mm, film thickness – 1.0 μm); sample size was 1 μl, detector temperature was 260 °C, injector temperature was 200 °C, and column temperature was 90 °C → 200 °C; helium was used as carrier gas (flow rate 5.0 ml·min⁻¹).

Analysis of protein degradation indicators

Selected indicators of CP degradation were analyzed in experimental silages. The TP content of silage was determined using 10% trichloroacetic acid. The following CP fractions were determined: soluble crude protein (SCP), neutral detergent-insoluble crude protein (NDICP), and acid detergent-insoluble crude protein (ADICP), according to the methods proposed by Licitra et al.²¹. Ammonia nitrogen content was determined by direct distillation using the 2100 Kjeltec Distillation unit (Foss Analytical A/S, Hilleröd, Denmark). Herbage samples were subjected to identical analyses.

Based on the results of the above analyses of CP fractions and NH₃–N content, CP was divided into the following fractions according to the Cornell Net Carbohydrate and Protein System (CNCPS v 6.5): PA1 – NH₃–N; PA2 – buffer-soluble true protein; PB1 – buffer-insoluble true protein (moderately degradable); PB2 – fiber-bound protein (slowly degradable); PC – indigestible protein. The proportions of CP fractions (CNCPS v 6.5) were calculated using the equations proposed by Higgs et al.²² and Van Amburgh et al.²³:where: – ammonia nitrogen (% CP), – buffer-soluble true protein (% CP), – buffer-insoluble true protein (moderately degradable) (% CP), – fiber-bound protein (slowly degradable) (% CP), – indigestible protein (% CP), – ammonia nitrogen (% SCP), – soluble crude protein (% CP), – crude protein (% DM), – neutral detergent-insoluble crude protein (% CP), – acid detergent-insoluble crude protein (% CP).

Statistical analysis

For data parametrization, the results of observations (dependent variables) were evaluated for normality of distribution by the Shapiro–Wilk test, and the homogeneity of variance was assessed by Levene’s test. Testing was carried out for the entire population of results for a given observation, the main effects, and each experimental treatment. The global effect(s) of a factor/factors and their interactions on each group of observations were determined by analysis of variance (ANOVA). In the next step, post-hoc multiple comparisons were performed using Tukey’s test and pairwise comparisons were performed using Dunnett’s test. The significance of differences between means in experimental treatments was determined at p = 0.05. Statistical calculations were performed and data were visualized using STATISTICA (Statsoft version 13.3, TIBCO Software Inc., Palo Alto, California, United States) and MATLAB 2021 (MATLAB Software, MathWorks, USA) software.

Results

Chemical composition of alfalfa herbage before ensiling

The chemical composition of alfalfa herbage before ensiling is presented in Table 1. Wilting induced an increase in the concentrations of DM, SCP, NDICP, and ADICP, an increase in the proportions of CP fractions PA1, PA2, and PB2, a decrease in TP content, and a decrease in the proportion of fraction PB1. In unwilted alfalfa herbage (0-h wilting), TP accounted for 79.04% CP; after a 12-h wilting, TP content decreased to 57.93% CP and, consequently, the proportion of fraction PB1 decreased from 64.15% CP in unwilted alfalfa herbage (0-h wilting) to 57.86% CP after a 12-h wilting.

Table 1. Chemical composition of alfalfa before ensiling.

Item	Herbage after a 0-h wilting	Herbage after a 12-h wilting
Dry matter, g kg−1	221.2	351.3
Crude protein, g kg−1 DM	214.8	220.1
TP, % CP	79.04	57.93
SCP, %CP	4.63	11.92
NDICP, %CP	3.80	6.68
ADICP, %CP	1.29	2.69
PA1, % CP	0.43	0.58
PA2, % CP	31.62	34.88
PB1, % CP	64.15	57.86
PB2, % CP	2.51	3.99
PC, % CP	1.29	2.69

DM, dry matter; CP, crude protein; TP, true protein; SCP, soluble crude protein; NDICP, neutral detergent-insoluble crude protein; ADICP, acid detergent-insoluble crude protein; PA1, ammonia nitrogen; PA2, buffer-soluble true protein; PB1, buffer-insoluble true protein (moderately degradable); PB2, fiber-bound protein (slowly degradable); PC, indigestible protein.

Fermentation parameters of silage

During the ensiling process, only 0-h wilting treatments were characterized by a rapid decrease in pH over the first 3 days, regardless of the amount of added dry ice, and this trend was maintained only in the silage made from unwilted alfalfa (0-h wilt) with the addition of 2 g of dry ice (Fig. 1a). In silages produced from unwilted alfalfa (0-h wilt) with the addition of 0 g and 0.5 g of dry ice, pH increased after three days of fermentation and then decreased again on day 15. Regardless of the amount of added dry ice, silages produced after a 12-h wilting were characterized by a somewhat slower decline in pH, and this trend was maintained only in the silage made after a 12-h wilting with the addition of 2 g of dry ice. After 30 days of fermentation, silages produced from unwilted alfalfa (0-h wilting) with the addition of 0.5 g, 1 g, and 2 g of dry ice had significantly lower pH values (p = 0.048) (Table 2).

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Fig. 1

Effect of dry ice addition and wilting on the rate of changes in pH (a) and the concentrations of lactic acid (b) and acetic acid (c) in alfalfa silage.

Table 2. Effect of dry ice addition and wilting on fermentation pattern and protein fractions in alfalfa silage on day 30.

Item	Dry ice (D)	0 g		0.5 g		1 g		2 g		SEM	p-value
	Wilting (W)	0 h	12 h	0 h	12 h	0 h	12 h	0 h	12 h		D	W	DxW
DM, g kg−1		234.3b	361.6a	228.1b	358.8a	229.8b	357.9a	228.6b	364.6a	5.915	0.084	0.009	0.015
CP, g kg−1 DM		228.4	233.7	232.6	217.3	229.1	222.9	238.4	219.2	0.866	0.090	0.560	0.149
Fermentation pattern, g kg−1 DM
pH		4.52ab	4.82a	4.25b	4.55ab	4.35b	4.59ab	4.24b	4.57ab	0.034	0.145	0.208	0.048
NH3–N		103.8abc	112.7a	95.2bc	80.4b	90.9bc	99.2abc	72.5bd	81.1b	3.243	≤ 0.001	0.019	≤ 0.001
LA		76.7bde	58.3bdf	103.2a	67.5bd	97.5abc	66.2bd	105.4a	93.4bc	2.391	0.418	0.001	0.016
AA		6.33abc	8.10a	5.69be	3.96bde	5.78be	4.01bde	0.31bdf	4.14bde	0.170	0.048	0.009	≤ 0.001
PA		0.37a	0.39a	0.27ab	0.41a	0.29ab	0.46a	0.01b	0.35a	0.012	0.698	0.080	≤ 0.001
BA		0.71a	0.01b	0.07b	0.02b	0.06b	0.02b	0.05b	0.02b	0.017	0.077	0.027	0.001
Et		0.01b	0.01b	0.01b	0.01b	0.01b	0.01b	0.37a	0.02b	0.047	0.012	0.060	≤ 0.001
SCFAs		7.42a	8.51a	6.05abc	4.40bde	6.15abc	4.52be	1.92bdf	4.53be	0.178	0.029	0.016	≤ 0.001
Protein fractions, % CP
TP		25.53bcd	29.79bcd	22.43bd	34.94bc	24.98bd	35.36bc	24.46bd	41.54a	0.718	0.027	0.001	0.014
SCP		60.08a	62.11a	59.45abc	59.26abc	56.79bcde	54.30bcde	52.19bde	43.49bdf	0.534	0.034	0.003	0.011
NDICP		10.07b	11.10ab	10.08b	11.56ab	10.91ab	10.62ab	10.87ab	11.68a	0.085	0.047	0.025	0.022
ADICP		7.44abc	7.39abc	7.56abc	7.97a	7.12bc	7.46abc	6.99bc	6.15bd	0.066	0.047	0.008	0.044
Protein fractions according to CNCPS, % CP
PA1		6.26abc	7.01a	5.66bde	4.77bd	5.17bde	5.38bde	3.78bdf	3.53bdf	0.023	0.002	0.021	0.027
PA2		53.82abc	55.10a	53.79abc	54.49a	51.62b	48.92bde	48.41bde	39.96bdf	0.463	0.017	0.019	0.028
PB1		29.85bd	26.79bd	30.48b	29.19bd	32.30b	35.08bc	36.93bc	44.83a	0.503	0.012	0.047	0.045
PB2		2.63bd	3.71bc	2.52bd	3.59bc	3.79bc	3.16b	3.83bc	5.53a	0.091	0.038	≤ 0.001	≤ 0.001
PC		7.44abc	7.39abc	7.56abc	7.97a	7.12bc	7.46abc	6.99bc	6.15bd	0.066	0.047	0.008	0.044

DM, dry matter; CP, crude protein; NH₃–N, ammonia nitrogen (g⋅kg⁻¹ N_total); LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; Et, ethanol; SCFAs, short-chain fatty acids; TP, true protein; SCP, soluble crude protein; NDICP, neutral detergent-insoluble crude protein; ADICP, acid detergent-insoluble crude protein; PA1, ammonia nitrogen; PA2, buffer-soluble true protein; PB1, buffer-insoluble true protein (moderately degradable); PB2, fiber-bound protein (slowly degradable); PC, indigestible protein. SEM, standard error of the mean. Values followed by the same superscript letters (a–f) are not significantly different at P ≤ 0.05.

The LA content increased most rapidly during the first three days of fermentation in the silage made from unwilted alfalfa (0-h wilting) with the addition of 1 g of dry ice, and it remained stable from day 7 of fermentation (Fig. 1b). In silages produced after a 12-h wilting with the addition of 1 g and 2 g of dry ice, LA concentration increased somewhat slower but still fast, and it also remained stable from day 7 of fermentation in these experimental treatments, and increased again in the silage made after a 12-h wilting with the addition of 2 g of dry ice between days 15 and 30 of fermentation. On day 30 of fermentation, LA concentration was significantly higher in silages produced from unwilted alfalfa (0-h wilting) (p = 0.016) (Table 2). In silages produced from alfalfa wilted for 12 h, the addition of 2 g of dry ice contributed to a significantly higher ultimate concentration of LA (p = 0.016).

The acetic acid concentration increased most rapidly during the first three days of fermentation, except in the silage made from unwilted alfalfa (0-h wilting) with the addition of 2 g of dry ice (Fig. 1c). In this experimental treatment, AA level was very low and remained stable throughout the ensiling process. In the remaining silages, AA concentration increased gradually until day 30 of fermentation; it reached the highest level in the silage made from alfalfa wilted for 12 h without dry ice addition (0 g) and lowest level in the silage produced from unwilted alfalfa (0-h wilting) with the addition of 2 g of dry ice (p ≤ 0.001) (Table 2). Butyric acid concentration was significantly highest in the silage made from unwilted alfalfa (0-h wilting) without dry ice addition (0 g) (Table 2). The remaining silages were characterized by similar BA levels after 30 days of fermentation (p = 0.001). On day 30 of fermentation, SCFA concentrations were significantly highest in silages produced from unwilted alfalfa (0-h wilting) and alfalfa wilted for 12 h without dry ice addition (0 g) (p ≤ 0.001).

Protein fractions of silage

True protein content decreased throughout the ensiling process, and the most rapid decrease was observed in silages produced after a 12-h wilting with the addition of 0 g, 0.5 g, 1 g, and 2 g of dry ice during the first three days of fermentation, and in the remaining silages during the first seven days of fermentation (Fig. 2a). In silages made from alfalfa wilted for 12 h, the decrease in TP content from day 3 of fermentation was slower, and slowest in the silage produced after a 12-h wilting with the addition of 2 g of dry ice. In the latter treatment, TP concentration was significantly highest on day 30 of fermentation (p = 0.014) (Table 2). An increase in the proportion of fraction PA1, which corresponds to the NH₃–N content of CP, was slowest during the first three days of fermentation in all silages excluding the silage made from unwilted alfalfa (0-h wilting) with the addition of 1 g and 2 g of dry ice (Fig. 2b). On successive days of fermentation, a considerable increase was noted in the proportion of fraction PA1, which stabilized on day 15 in silages produced from unwilted alfalfa (0-h wilting) and alfalfa wilted for 12 h with the addition of 2 g of dry ice. These experimental treatments were characterized by the lowest ultimate concentration of PA1 (p = 0.027) (Table 2). The proportion of fraction PA1 decreased in the silage made from unwilted alfalfa (0-h wilting) with the addition of 1 g of dry ice between days 15 and 30 of fermentation (Fig. 2b).

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Fig. 2

Rate of changes in the proportion of true protein (a) in total crude protein and the proportions of fractions PA1 (b), PA2 (c), PB1 (d), PB2 (e), and PC (f) in total crude protein in alfalfa silage.

The proportion of fraction PA2 varied in all silages on successive days of fermentation (Fig. 2c). The smallest and slowest increase in the proportion of fraction PA2 was noted in the silage made after a 12-h wilting with the addition of 2 g of dry ice. In this experimental treatment, PA2 concentration increased only during the first three days of fermentation. In turn, the silage produced from unwilted alfalfa (0-h wilting) with the addition of 2 g of dry ice was characterized by a minor increase in the proportion of fraction PA2 until day 3 of fermentation, followed by a considerable increase in PA2 concentration between days 7 and 15 of fermentation. On day 30 of fermentation, the proportion of fraction PA2 was lowest in the silage made after a 12-h wilting with the addition of 2 g of dry ice (Table 2).

The proportion of fraction PB1 decreased in all silages on successive days of fermentation (Fig. 2d). The slowest decrease in PB1 concentration was observed in the silage produced from alfalfa wilted for 12 h with the addition of 2 g of dry ice. In the silage made from unwilted alfalfa (0-h wilting) with the addition of 2 g of dry ice, the proportion of fraction PB1 decreased slowly until day 7 of fermentation, then decreased considerably and remained stable between days 15 and 30 of fermentation. The proportion of fraction PB1 was lowest in the silage produced after a 12-h wilting without dry ice addition (0 g) (p = 0.045). The smallest increase in the proportion of fraction PB2 was noted in the silage made from alfalfa wilted for 12 h with the addition of 2 g of dry ice (Fig. 2e); after 30 days of fermentation, this silage had the highest concentration of fraction PB2 (Table 2). The greatest decrease in the proportion of fraction PB2 was found in the silage produced after a 12-h wilting with the addition of 0.5 g of dry ice during the first three days of fermentation (Fig. 2e).

The proportion of fraction PC represented by ADICP increased throughout fermentation (Fig. 2f). The slowest increase in the percentage of PC was observed in the silage made from alfalfa wilted for 12 h with the addition of 2 g of dry ice. In turn, the proportion of this fraction increased most rapidly in the silage produced after a 12-h wilting with the addition of 0.5 g of dry ice during the first three days of fermentation, a slower increase was observed until day 7 and between days 15 and 30 of fermentation. Silages made from unwilted alfalfa (0 h) with the addition of 1 g of dry ice and from alfalfa wilted for 12 h with the addition of 1 g of dry ice were characterized by a slow increase in the proportion of fraction PC during the first three days of fermentation, followed by its stable level until day 15 and a minor increase until day 30 of fermentation. On day 30 of fermentation, the percentage of fraction PC was lowest in the silage made after a 12-h wilting with the addition of 2 g of dry ice (Table 2).

Discussion

The first proteolytic changes occurred during the wilting of alfalfa herbage prior to ensiling, as indicated by a decrease in TP content and an increase in SCP content¹². In a study by Guo et al.⁸, TP content was 84.70% CP in fresh alfalfa and 82.90% CP in alfalfa wilted for 10 h. In another experiment²⁴, the TP content of fresh alfalfa herbage was 70.2% CP, and it decreased to 64.2% CP after a 36-h wilting; the latter value is higher than that noted in this study.

Significant differences between TP levels in fresh and wilted forage could be due to the different duration and rate of the wilting process⁸, i.e. the different rate of moisture loss by plant cells, affected by variable environmental conditions, including temperature, humidity, and air flow. Considerable effects are also exerted by plant material characteristics, including interspecific and inter-varietal differences such as the leaf/stem ratio and maturity stage at harvest²⁵. According to Cavallarin et al.²⁶, mechanical conditioning may limit the proteolysis and catabolism of essential amino acids by reducing the field wilting time.

The rapid decline in pH during the first three days of alfalfa ensiling, observed in this study, was also reported by Li et al.²⁷ and Wang et al.²⁸. In the current study, already the addition of 0.5 g of dry ice, as well as its larger amounts (1 g and 2 g), to unwilted alfalfa speeded up the acidification of the ensiled forage mass over the first three days. This could be due to the higher moisture content of plant cells, which stimulated the growth of some bacteria of the genus Pedicoccus, which usually proliferate rapidly and promote LA fermentation in the early stage of ensiling, leading to an initial drop in silage pH²⁸.

In the present study, the ultimate pH on day 30 of fermentation was within the range recommended for alfalfa with a DM content of 30–35% (pH = 4.3–4.5)²⁹. Rapid initial acidification is the key to controlling the growth of enterobacteria and clostridia³⁰. Agarussi et al.³¹ found that the counts of enterobacteria decreased rapidly when the pH of alfalfa silage dropped to 4.74 during the first three days of fermentation, which was confirmed by Wang et al.²⁸. The inhibition of enterobacterial growth can be attributed to acidic conditions (pH below 5.0) that develop in silage³².

Similarly to this study, also in the experiment conducted by Li et al.²⁷ LA content increased until day 7 and then until day 15 of fermentation, and the noted values were comparable to or higher than those recommended for LA in alfalfa silage (60–80 g kg⁻¹ DM) by Kung et al.²⁹. The lower LA content of wilted alfalfa silage was due to the fact that the availability of metabolic water for the growth of LA bacteria was reduced with increasing DM concentration³³.

In the present study, AA concentration was lower than the values reported by Li et al.²⁷ (11.4 g kg⁻¹ DM) and Kung et al.²⁹ (20–30 g kg⁻¹ DM). A moderate AA content of silage may be beneficial because AA inhibits yeast growth, thus improving the stability of silage exposed to air. Actually, silages with a low concentration of AA can be unstable. Similarly, LA and AA concentrations are usually inversely proportional to silage DM content²⁹, but this relationship was not observed in this study.

The quality of fermentation may affect proteolysis in ensiled alfalfa because proteases are sensitive to changes in the pH of biomass, and deamination may be promoted by microbial activity. Furthermore, fermentation products such as LA and AA may reduce the microbial activity related to proteolysis³⁴. The ultimate pH, and the concentrations of total and individual acids on day 30 of fermentation indicate that LA bacteria dominated in all silage treatments. In the current experiment, BA content was below 0.71 g kg⁻¹ DM, which points to very low activity of Clostridium spp.²⁹. The analyzed fermentation parameters indicate that homofermentation, with LA as the main product, predominated in alfalfa silages with the addition of dry ice³⁰.

The rate of changes in TP concentration, observed in the present study, is consistent with that reported by Li et al.²⁷, Guo et al.³⁵ and Yuan et al.³⁶. A linear decrease in TP content and an increase in the proportion of fraction PA1 were indicative of reduced proteolysis in the silage made after a 12-h wilting with the addition of 2 g of dry ice, followed by silages produced after a 12-h wilting and treated with 1 g and 0.5 g of dry ice. In comparison with the control silage, the rate of proteolysis was lower than expected in the above treatments. Although the rate and extent of proteolytic processes in alfalfa silage treated with dry ice have not been described in the literature, the present study confirmed certain patterns of changes in CP fraction composition during the ensilage of wilted alfalfa with the addition of dry ice.

The NH₃–N production, which is one of the indicators of CP degradation, can be stimulated by the activity of Enterobacteriaceae and Clostridium spp.²⁸. In the current experiment, NH₃–N concentration in alfalfa silages was adequate and confirmed their high quality²⁹. The above findings suggest that the decrease in silage pH was rapid and sufficient due to the addition of dry ice.

Microbial activity in silage can be estimated based on NH₃–N content because amino acids are broken down to NH₃–N during deamination by bacteria producing BA, AA, and LA¹². In addition, low NH₃–N content is usually related to low pH in alfalfa silage³⁷. In the present study, the proportion of NH₃–N changed dynamically, suggesting that deamination occurred throughout the ensiling process in unwilted alfalfa (0-h wilting), and the extent of this process decreased with increasing amounts of dry ice added to silages made after a 12-h wilting.

In the current experiment, CP quality was evaluated and the ruminal degradability of CP fractions was predicted according to the CNCPS²². The obtained results were consistent with those reported by Nadeau et al.³⁸ who noted a decrease in TP concentration and an increase in the proportion of PC after ensiling. The fact that TP concentration increased in response to increasing quantities of dry ice added to alfalfa ensiled after a 12-h wilting indicates that the efficiency of N utilization by ruminants can be improved at higher addition levels of dry ice. Nguyen et al.³⁹ found a negative correlation between the in vitro ruminal degradability of buffer-insoluble N and urinary N excretion in ruminants (sheep).

The proportion of fraction PB1 in the treatments characterized by the highest extent of proteolysis, i.e. in silages produced from unwilted alfalfa herbage (0-h wilting), was similar to the values reported by Li et al.²⁷ for alfalfa silage. According to the literature data, the proportion of fraction PB1 in alfalfa silage typically ranges from 14.60% CP⁸ to 27.00% CP⁴⁰, which is much lower than the values noted in silages treated with 1 g and 2 g of dry ice, regardless of the degree of wilting. The wide ranges of PB1 fractions presented in the literature, as well as the high values in our own studies according to Sousa et al.⁴⁰ are due to environmental variability rather than to differences in alfalfa cultivars.

According to Hymes-Fecht et al.⁴¹, alfalfa silage is usually characterized by the lowest proportion of fraction PB2. Similar observations were made by Grabber and Coblentz⁴², and Hartinger et al.⁴³, who found that PB2 accounted for 0.00–0.20% CP in alfalfa silages regardless of the degree of wilting. In the present study, PB2 was also the smallest or the second smallest (after PA1) CP fraction in alfalfa silages²¹. Similar proportion of fraction PB2 in the total CP of alfalfa silage was reported by Guo et al.⁸ (3.26% CP) and Sousa et al.⁴⁰ (3.80% CP), whereas a higher value was noted by Li et al.²⁷ (7.52% CP). Such a low percentage of fraction PB2 was indirectly determined by the proportion of fraction PC²¹.

In the current study, the proportion of fraction PC in alfalfa silages was in the middle of the wide range of values (1.71% to 10.50% CP) described in the literature^{40, 41–42}. An even higher proportion of fraction PC in ensiled alfalfa was reported by Guo et al.⁸ (12.30% CP) and Li et al.²⁷ (14.80% CP). The lower proportion of fraction PC observed in this study could be associated with potentially higher CP digestibility⁴⁴. The range of PC fractions is primarily influenced by the level of heat accumulation during ensiling of plants^8,12. Under the influence of temperature and/or contact with oxygen, the carbonyl group of the reducing sugar may condense with the free amino group of the amino acid, peptide or protein, resulting in an increase in the share of the PC fraction¹². Guo et al.⁸ observed an increase in the share of the PC fraction in alfalfa silage treated with tannic acid, which they explained by the formation of tannic acid-protein complexes, which also indicates the role of the ensiling additive used in the concentration of the PC fraction.

Fraction PB1 is partially hydrolyzed in the rumen and partially passes to the lower sections of the intestine, whereas a high percentage of faction PB2 escapes degradation in the rumen²². An analysis of fraction PB2 suggests that the proportion of TP passing to the lower sections of the intestine would increase with increasing amounts of dry ice added to wilted alfalfa during ensilage. Fraction PC contains Maillard reaction products, tannin-protein complexes, and fiber-bound protein, which cannot be degraded by ruminal bacteria²¹. Similar observations were made by Guo et al.⁸ in whose study ensiled alfalfa had a higher proportion of fraction PC than fresh herbage, most likely due to heat accumulation. In the present study, the significantly lowest proportion of fraction PC in the alfalfa silage made after a 12-h wilting with the addition of 2 g of dry ice, relative to the control treatment, points to a beneficial influence of dry ice and wilting, leading to higher CP availability. An analysis of the proportions of all CP fractions suggests that ensiling alfalfa after a 12-h wilting with the addition of 2 g of dry ice could reduce N excretion and improve the efficiency of N utilization in the rumen⁴⁵.

Conclusions

The present study demonstrated that CP proteolysis during ensilage was limited when alfalfa herbage was wilted for 12 h prior to ensiling and 2 g of dry ice was added to the ensiled forage mass without compromising the quality of fermentation in mini silos on a laboratory scale. Wilting and the addition of 2 g of dry ice, a linear decrease was noted in pH, TP content, and the proportions of fractions PA2, PB1, and PB2, whereas the concentrations of LA and AA, and the proportion of fraction PC increased gradually and observed a significantly lower concentration of NH₃–N and significantly lower proportions of fractions PA1 and PA2, compared with the control silage. In the future, alfalfa silage with the addition of dry ice should be produced on the industrial scale, and the efficiency of N utilization in dairy cows fed alfalfa silage treated with dry ice should be analyzed.

Acknowledgements

This research was funded in whole or in part by the National Science Centre, Poland Grant No. 2021/41/N/NZ9/01881. For the purpose of Open Access, the author has applied a CC-BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission.

Author contributions

Conceptualization, M.B-S., C.P.; methodology, M.B-S., C.P.; software, M.B-S., I.B.; validation, M.B-S., C.P., I.B.; formal analysis, M.B-S., I.B., SW.P.; investigation, M.B-S., C.P., I.B.; resources, M.B-S., C.P.; data curation, M.B-S., I.B.; writing-original draft preparation, M.B-S., C.P., I.B.; writing-review and editing, M.B-S., C.P., I.B., SW.P.; visualization, M.B-S., I.B.; supervision, M.B-S., C.P.; project administration, M.B-S.; funding acquisition, M.B-S., C.P.

Data availability

The datasets generated and analyzed during the current study have been partially deposited in the UWM Knowledge Base (DOI: 10.31648/UWMfaae34330dfe41d18b4cd511ee16d46f), as well as being available from the author corresponding to a reasonable request. Correspondence and requests for materials should be addressed to M.B-S. at [email protected].

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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