Effects of Storage Time and Temperature on the Fermentation Characteristics of Rumen Fluid from a High-Forage Diet
Jiangxi Province Key Laboratory of Animal Nutrition and Feed, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China
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Author to whom correspondence should be addressed.
Agriculture 2024, 14(9), 1481; https://doi.org/10.3390/agriculture14091481 (registering DOI)
Submission received: 22 July 2024
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Revised: 24 August 2024
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Accepted: 29 August 2024
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Published: 1 September 2024
(This article belongs to the Section Farm Animal Production)
Abstract
:The objective of this experiment was to investigate the effects of storage temperature and preservation time on the fermentation characteristics of rumen fluid collected from six Hu sheep fed a high-forage diet. The storage temperatures were set at −80 °C and −20 °C, and the preservation times were labelled as follows: 0 d (fresh rumen fluid, D0), 7 d (D7), 14 d (D14), 30 d (D30), 60 d (D60), 120 d (D120), and 240 d (D240). A repeated-measures design was applied to analyze the fermentation characteristics of rumen fluid across each preservation time point and storage temperature. The results showed that storage temperature had no significant effects on pH value, ammonia nitrogen (NH3-N), microbial protein (MCP), and volatile fatty acid (VFA) concentration and proportion (p > 0.05). Specifically, the pH value increased on D7, D14, and D60 (p < 0.05), while the MCP concentration decreased on D7, D14, D30, D120, and D240 but increased on D60 (p < 0.05), and the concentration of NH3-N decreased on D14 (p < 0.05). The acetate concentration increased on D30, while the concentrations of propionate, butyrate, valerate, and total VFA increased on D60 (p < 0.05). The concentration of isovalerate decreased on D60, and isobutyrate and branched-chain VFA concentrations decreased on D120 (p < 0.05). The proportion of acetate increased on D30, while the butyrate and valerate proportions increased on D60, and the proportions of isovalerate, isobutyrate, and branched-chain VFA decreased on D60 (p < 0.05). For rumen fluid from a high-forage diet, the storage temperature (−80 °C and −20 °C) did not affect its fermentation characteristics, suggesting that rumen fluid could be preserved at −80 °C or −20 °C without altering its fermentation characteristics. The fermentation parameters of rumen fluid changed significantly after 7 days of preservation; hence, it is recommended to finish determining the fermentation parameters of rumen fluid within 7 days of collection. The concentrations and proportions of most VFA changed after 30 days of preservation; therefore, it is recommended that VFA determination should be completed within 30 days of rumen fluid collection.
1. Introduction
Rumen fluid consists of components derived from the degradation and utilization of dietary nutrients by rumen microorganisms, such as volatile fatty acids (VFA), ammonia nitrogen (NH3-N), and microbial proteins (MCP) [1,2]. These components reflect the degradation and utilization of dietary nutrients in the rumen [2]. Additionally, rumen fluid is rich in microorganisms and can serve as a culture medium for in vitro rumen fermentation [3,4,5]. For rumen fluid collected during ruminant nutrition experiments, it is not always possible to complete the analysis of all parameters within a short period. However, it is not yet clear how long rumen fluid can be stored while still accurately reflecting feed degradation and utilization in the rumen. As a culture medium for in vitro rumen fermentation tests, the duration of storage that maintains the rumen fluid’s effectiveness is crucial for enhancing the repeatability of in vitro experiments. Therefore, it is necessary to clarify the dynamic fermentation characteristics of rumen fluid at different storage time points to determine the appropriate time for sample analysis and assess whether it can be used as a culture medium for in vitro rumen fermentation tests.
In vitro rumen fermentation studies typically require multiple repetitions to enhance the credibility of the results. Differences in the physicochemical properties of rumen fluid between batches can affect the outcomes, suggesting that using the same rumen fluid for these tests is theoretically more convincing. However, obtaining the same rumen fluid at different time points or from different individual animals presents challenges and is primarily influenced by factors such as diet type, feed intake, and collection methods [6,7,8,9]. Preserving rumen fluid under appropriate conditions is a potentially feasible method of obtaining consistent rumen fluid [10]. Some researchers have studied the feasibility of using rumen fluid under different storage conditions as a culture medium for in vitro rumen fermentation tests. These studies have found that rumen fluid can be used as a culture medium for in vitro rumen fermentation when stored at 4 °C, −20 °C, and −80 °C or in liquid nitrogen [6,7,8,9]. Storing rumen fluid at 18 °C for 48 h or at 40 °C for 5 h did not affect its use in in vitro studies of feed degradation characteristics [11,12]. However, the aforementioned studies focused on the short-term (within one month) storage of rumen fluid samples, and the feasibility of using rumen fluid as an in vitro rumen fermentation culture medium after longer storage times remains unclear. The fermentation characteristics of rumen fluid reflect the activity of microorganisms, which is closely related to the effectiveness of in vitro cultures [6,7,8,9]. Therefore, the feasibility of using rumen fluid as a culture medium for in vitro fermentation tests can be indirectly judged from its fermentation characteristics.
In the actual process of sample analysis, it typically takes several weeks to complete all determinations on rumen fluid. During these weeks, the key question is whether there are significant differences in the fermentation characteristics between stored and fresh rumen fluid. This difference determines whether the analysis results can truly represent the degradation and utilization of feed in the rumen. A few studies have reported changes in the physicochemical properties of rumen fluid over short storage periods. Fabro et al. [13] found that the pH value and VFA content of rumen fluid stored at 4 °C for 96 h were not different from fresh rumen fluid, while the NH3-N content increased after 48 h of storage. Qiu et al. [14] observed changes in the physicochemical parameters of rumen fluid fed with a high-concentrate diet after storage at −20 °C for 60 days. However, Martin et al. [15] found that the physicochemical properties of rumen fluid varied with the storage time and were related to the type of diet. Therefore, it is necessary to explore the dynamic changes in the fermentation characteristics of rumen fluid from various diet types during storage.
Currently, laboratories use −20 °C or −80 °C for long-term sample preservation [16]. The storage at −80 °C has a higher maintenance cost. Due to the large volume of samples used for in vitro fermentation tests [7], the limited space in −80 °C freezers makes it difficult to meet practical needs. If the fermentation characteristics of rumen fluid stored at −20 °C or −80 °C do not differ significantly, it would be possible to store rumen fluid in −20 °C freezers, thereby saving considerable space in −80 °C freezers. Based on these considerations and research progress, this experiment started with fresh rumen fluid as the initial sample. The goal was to explore the dynamic changes in its fermentation characteristics under different storage temperatures over time. The aim is to provide data references for the recommendation of the appropriate time for parameter determination and the ideal storage conditions of rumen fluid for in vitro rumen fermentation tests. We hypothesized that the fermentation characteristics of rumen fluid would vary with the storage time and temperature, and these patterns of change would be related to specific indicators.
2. Materials and Methods
2.1. Source of Rumen Fluid
The animal care and welfare protocols were approved by the Committee for the Care and Use of Experimental Animals at Jiangxi Agricultural University, with the approval number JXAULL-2021036. Rumen fluid was obtained from six Hu sheep fed a high-forage diet; the choice of six sheep was based on previous studies regarding the number of donors greater than four being adequate for in vitro fermentation tests and rumen fluid preservation investigations [7,13]. The dietary ingredient and chemical composition are detailed in Table 1. After being fed the aforementioned diet for three months, the six sheep were humanely sacrificed in strict accordance with the operating procedures of livestock and poultry slaughtering—sheep and goat (NY/T 3469-2019). The process included preslaughter inspection, stunning, bleeding, evisceration, cleaning, chilling, meat inspection, and packaging to ensure animal welfare and product safety. Following the recommendations of Mulder et al. [17], ruminal contents were collected from the dorsal, central, and ventral regions of rumen at the time of slaughter. The rumen was immediately removed and its contents were mixed evenly to ensure the representativeness of samples. The collected ruminal contents were then passed through four layers of gauze to obtain fresh rumen fluid samples, which were designated as the 0-day samples. It should be noted that the decision to slaughter six sheep was made due to the necessity to procure a sufficient volume of rumen fluid for a series of in vitro batch fermentation tests, as well as for the current experiment. Throughout this procedure, we strictly adhered to stringent regulations to safeguard animal welfare.
2.2. Experimental Design
A total of six Hu sheep, with similar body weight (17.84 ± 0.43 kg) and age (89.67 ± 1.21 day), were housed individually in separate pens for three months. During this period, they had free access to feed and clean water. Rumen fluid collected from each sheep was evenly mixed to create a representative sample for that individual. Consequently, six rumen fluid samples were finally obtained in total and divided into dozens of cryogenic tubes. These samples were stored at −80 °C and −20 °C, with preservation time designated on 0 days (D0), 7 days (D7), 14 days (D14), 30 days (D30), 60 days (D60), 120 days (D120), and 240 days (D240), where D0 represents fresh rumen fluid. The fermentation characteristics of rumen fluid measured in this experiment included pH value, NH3-N, MCP, and VFA. Each parameter at different time points was measured by the same technician. The thawing method for rumen fluid is as follows: first, the samples were placed in an environment of 0 °C to 4 °C to melt, then placed in a water bath. The water bath was set to increase the temperature by 1 °C every 3 min, ultimately reaching 39 °C to minimize temperature shock [7].
2.3. Parameter Determination
The parameters measured in this experiment include pH value, NH3-N, MCP, and VFA. The pH value was measured using a portable pH meter (testo 206, testo AG, Schwarzwald, Germany) immediately after the rumen fluid was collected or once the thawed sample reached 39 °C. NH3-N was determined using the phenol-hypochlorite colorimetric method [18], and MCP was analyzed using the Folin-Phenol method as described by Makkar et al. [19]. The colorimetric analysis for the above two methods was performed using a microplate reader (MK3, Thermo Fisher Scientific, Waltham, MA, USA). The VFA detected includes acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate. The sum of these six VFAs is referred to as the total volatile fatty acids (TVFA). The sum of isobutyrate, valerate, and isovalerate constitutes the branched-chain volatile fatty acids (BCVFA) [20]. The ratio of a single volatile fatty acid to TVFA is the proportion of that VFA, expressed as a percentage (%). The determination of VFA was performed using a gas chromatograph (Shimadzu GC2014, Shimadzu Corporation, Kyoto, Japan) equipped with a 30 m capillary column (Rtx-Wax, 0.25 mm ID × 0.25 μm film, Restek, Evry, France) and flame ionization detector (FID-2014, Shimadzu Corporation, Kyoto, Japan). The injection volume and injector temperature were kept at 0.4 μL and 220 °C, respectively. The oven program proceeded as follows: initially 110 °C for 30 s, up to 120 °C in 3 min and maintain for 4 min, and continue to 150 °C at the constant rate of 10 °C/min. The split ratio was maintained at 20:1 and flow rate was kept at 2.5 mL/min. The analytical standard used for quantification was procured from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The identity of each VFA was verified based on relative retention time and the concentration of each VFA was calculated using the external standard method [21].
2.4. Statistical Analysis
All data were analyzed using the general linear model of repeated measures in SPSS software (version 20, IBM Corporation, Armonk, NY, USA). The statistical model is formulated as follows: Yijk = μ + Ti + Pj + (TP)ij + Rk + eijk, where Yijk represents the dependent variable, μ is the overall mean, Ti is the fixed effect of storage temperature (i = 2), Pj is the fixed effect of preservation time (j = 7), (TP)ij is the interaction effect between storage temperature and preservation time, Rk is the random effect, and eijk is the random error. To facilitate comparison of fermentation characteristics between rumen fluid at each time point after freezing preservation and fresh rumen fluid, we selected differences at each time point for analysis. Additionally, pairwise comparisons of the p value were conducted between D7, D14, D30, D60, D120, D240, and D0, denoted as P1*. The significance level for these tests is set at p < 0.05.
3. Results
3.1. Rumen Fermentation Parameter
The dynamic changes in the fermentation parameters of rumen fluid under two storage temperatures over time are listed in Table 2. The storage temperature had no significant effect on the pH value, MCP, and NH3-N concentrations (p > 0.05). Furthermore, no interaction effect between storage temperature and preservation time on these three rumen fermentation parameters was observed (p > 0.05). The pH value increased on D7, D14, and D60 (p < 0.05). The concentration of MCP decreased on D7, D14, D30, D120, and D240, but it increased on D60 (p < 0.05). The concentration of NH3-N decreased at D14 (p < 0.05) and, although it increased numerically at D60, the difference was not significant (p > 0.05).
3.2. VFA Concentration
The dynamic changes in VFA concentration of rumen fluid under two storage temperatures over time are shown in Table 3. The storage temperature did not significantly affect the concentration of individual VFA, TVFA, or BCVFA (p > 0.05). Additionally, no interaction effect between storage temperature and preservation time on these VFA concentrations was observed (p > 0.05). The concentrations of all detected VFA changed with preservation time (p < 0.05), with specific trends as follows: the concentration of acetate increased on D30, D60, and D120; the propionate, butyrate, and TVFA concentrations increased on D60 and D120; the concentrations of isobutyate and BCVFA decreased on D120 and D240, while the concentrations of isovalerate and valerate exhibited a decrease and an increase after 60 days of preservation, respectively.
3.3. VFA Proportion
The dynamic changes in VFA proportions of rumen fluid under two storage temperatures over time are detailed in Table 4. The storage temperature did not significantly affect the proportions of individual VFA, BCVFA, or the acetate-to-propionate ratio (p > 0.05). Furthermore, no interaction effect between storage temperature and preservation time was observed for these parameters (p > 0.05). Preservation time affected the proportions of isobutyrate, butyrate, isovalerate, valerate, and BCVFA (p < 0.05). Specifically, the proportions of isobutyrate, isovalerate, and BCVFA decreased after 60 days of preservation, whereas the proportion of butyrate increased on D60 and D120. Additionally, the valerate proportion increased after 60 days of preservation.
4. Discussion
4.1. Dynamic Changes in Fermentation Parameters of Rumen Fluid as Preservation Time and Temperature Varied
The rumen pH value is an important indicator of the rumen fermentation environment, typically ranging between 5.5 and 7.5 [22]. In conventional sampling and testing processes, the rumen pH value should be measured immediately after the rumen is removed or the rumen fluid is extracted [23,24,25]. This experiment revealed that the rumen fluid underwent obvious changes after being preserved for 7 days, and there was no consistent pattern of continuous increment or decrement with the extension of preservation time. This suggests that the rumen pH value should be determined using fresh rumen fluid. Further exploration is needed to determine the changes in rumen pH value at shorter time intervals, which will help assess the rationality of the current sampling routines. MCP is an important source of protein and amino acids for ruminants, and its concentration is closely related to the nitrogen and carbohydrate content in the diet, as well as the composition and activity of rumen microorganisms [26]. This experiment found that the MCP concentration decreased sequentially after being stored for 7, 14, and 30 days but increased when stored for 60 days and then decreased. Low-temperature freezing conditions may damage cell membranes and cause DNA denaturation, reducing the activity of rumen microorganisms [27]. This could lead to a decrease in the MCP concentration with the extension of storage time within 60 days. As freezing time extends, the protein in the rumen fluid is hydrolyzed into ammonia [28]. This ammonia, along with the energy produced by the hydrolysis of carbohydrates, synthesizes more amino acids and MCP, which could explain the increase in MCP concentration when stored for 60 days. However, as the protein and carbohydrates in the rumen fluid are consumed, the synthesis of MCP shows a decreasing trend. NH3-N is an intermediate product of protein degradation in the diet and a nitrogen source for microbial growth and reproduction; therefore, the concentration of NH3-N can be considered as a dynamic balance between the degradation yield of dietary protein and the utilization of the nitrogen source by microorganisms [29]. The NH3-N concentration remains stable if the rumen fluid is acidified when stored at −70 °C for 22 days [30]. This study found that the NH3-N concentration decreased when the rumen fluid was stored for 14 days, which may be due to the lack of acidification treatment. Previous study have revealed that the NH3-N concentration increased after being stored for 65 days at −20 °C [28]. However, in this experiment, the NH3-N concentration only increased numerically after being stored for 60 days (14.43 vs. 13.41 mg/100 mL), which may be due to insufficient hydrolysis of protein in the rumen fluid by rumen microorganisms.
Considering all rumen fermentation parameters detected in this experiment, no difference was observed between the two storage temperatures of −20 °C and −80 °C. Most parameters exhibited changes after a 7-day storage period, with particularly notable alterations observed after 60 days of storage. At this time point, both the pH value and MCP concentration increased, and the concentration of NH3-N showed a numerical increase. These findings suggest that the analyses of rumen fermentation parameters should ideally be completed within 7 days post-collection of rumen fluid.
4.2. Dynamic Changes in VFA Concentration and Proportion of Rumen Fluid as Preservation Time and Temperature Varied
VFA is the main form of energy utilization in ruminants, serving as the final product of feed degradation, and affects the production efficiency and product quality of ruminants [29,31,32]. Takizawa et al. [33] found that, while the concentration of VFA remained stable when the rumen fluid was stored at 4 °C for 2 days, an increase was observed when the rumen fluid was stored at 20 °C or 35 °C for the same period. In this study, the storage temperature of −20 °C and −80 °C both maintained the rumen fluid in a frozen state. Given the similar cold shock response of rumen microorganisms to low temperature, there was no significant temperature effect on the concentration of VFA. Rumen microorganisms exhibit a cold shock response under low-temperature conditions, which is characterized by changes in the synthesis of cell membrane lipids and proteins [13]. This response can enhance sugar metabolism, thereby leading to increased VFA synthesis [13]. Furthermore, the rumen fluid contains feed residues, and the indigestible substances within these residues gradually degrade into sugar, VFA, and amino acids as preservation time extends [33]. This degradation process ultimately results in an increase in VFA concentration. A previous study has shown an increase in VFA within 2 days at 20 °C and 35 °C [33]. However, in this study, an increase in propionte, butyrate, valerate, and TVFA concentrations was observed after 60 days of preservation. This discrepancy may be attributed to the slower degradation rate of indigestible substances by rumen microorganisms under frozen conditions. As for BCVFA, its concentration decreased after 60 days of preservation, likely because it primarily originates from the fermentation of dietary protein [34]. The degradation products of protein are used to synthesize MCP [35], which corresponds to the observed increase in MCP concentration after 60 days of preservation. Consistent with the concentration of VFA, storage temperature did not affect the proportions of all VFA. Furthermore, the preservation time point at which changes in these proportions occur corresponds to the time point at which changes in VFA concentration occur.
Having taken into account both the concentration and proportion of VFA, significant changes in these parameters were observed after the rumen fluid was stored for 30 days. The current storage temperature of −80 °C and −20 °C did not affect the concentration and proportion of VFA. These findings indicate that VFA analysis should be finished within 30 days following the collection of rumen fluid.
5. Conclusions
For rumen fluid collected from high-forage diet feeding sheep, storage at −80 °C and −20 °C did not affect rumen fermentation characteristics. However, the pH value and MCP concentration exhibited significant changes after 7 days of storage, while the NH3-N concentration changed after 14 days. Additionally, most of the VFA concentration and proportion underwent significant alterations after 30 days of storage. To enhance the representativeness of rumen fluid samples, it is recommended that the determination of rumen fermentation parameters should be completed within 7 days post-collection. Furthermore, the analysis of VFA should be completed within 30 days. These recommendations will contribute to optimizing determination time and storage condition for rumen fluid analysis in the ruminant nutrition research.
It should be recognized that the current results are based on rumen fluid from animals fed with a diet high in low-quality roughage. Further research is needed to establish storage condition recommendations for diets rich in high-quality roughage or high-concentrate diets. Furthermore, incorporating multi-omics technologies could offer a more in-depth mechanistic analysis for exploring rumen fluid preservation conditions, which may be an enticing future perspective.
Author Contributions
Conceptualization, Q.Q.; methodology, Q.Q. and M.Q.; validation, C.F., M.Q., and K.O.; formal analysis, C.F. and Q.Q.; investigation, C.F. and Q.Q.; data curation, Q.Q. and M.Q.; writing—original draft preparation, C.F. and Q.Q.; writing—review and editing, Q.Q.; visualization, Q.Q. and M.Q.; supervision, Q.Q.; funding acquisition, Q.Q. All authors have read and agreed to the published version of the manuscript.
Funding
This research was supported by Jiangxi Provincial Natural Science Foundation, 20232BAB215051; National Natural Science Foundation of China, 32260861 and 32160807; and China Agriculture Research System of MOF and MARA, CARS-37.
Institutional Review Board Statement
The animal study protocol was approved by the Committee for the Care and Use of Experimental Animals at Jiangxi Agricultural University (approval number JXAULL−2021036).
Data Availability Statement
All data are available in section.
Acknowledgments
We would like to express our thanks to Kairong Li and Xinfeng Chen for their selfless support in the accommodation and animal provisions.
Conflicts of Interest
The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
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Table 1.
Ingredients and chemical composition of diet for donor sheep.
| Ingredient | % of DM | Chemical Composition | Value |
|---|---|---|---|
| Peanut straw | 68.58 | Metabolizable energy, Mcal/kg | 1.93 |
| Corn | 11.22 | Crude protein, % | 14.64 |
| Soybean meal | 11.17 | Neutral detergent fiber, % | 40.67 |
| Wheat bran | 4.65 | Acid detergent fiber, % | 29.87 |
| Calcium hydrogen phosphate | 0.15 | ||
| Sodium bicarbonate | 0.24 | ||
| Salt | 0.49 | ||
| Premix 1 | 3.50 | ||
| Total | 100.00 |
Note: 1 Premix provided the following per kg of DM: 1400 mg of Fe, 1200 mg of Zn, 250 mg of Cu, 900 mg of Mn, 100,000 IU of vitamin A, 27,000 IU of vitamin D3, and 800 IU of vitamin E.
Table 2.
Dynamic changes in the fermentation parameters of rumen fluid with preservation time at two storage temperatures.
Table 2.
Dynamic changes in the fermentation parameters of rumen fluid with preservation time at two storage temperatures.
| Item | Storage Temperature | Preservation Time 1 | SEM 3 | p-Value 4 | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| D0 | D7 | D14 | D30 | D60 | D120 | D240 | Time | Temperature | Interaction | |||
| pH value | −80 °C | 6.93 | 7.00 | 7.00 | 6.95 | 6.99 | 6.98 | 6.84 | ||||
| −20 °C | 6.93 | 7.06 | 7.14 | 6.93 | 7.06 | 7.08 | 7.03 | 0.351 | 0.002 | 0.877 | 0.164 | |
| Average | 6.93 | 7.03 | 7.07 | 6.94 | 7.03 | 7.03 | 6.93 | |||||
| P1* 2 | 0.003 | 0.001 | 0.804 | 0.040 | 0.059 | 0.836 | ||||||
| MCP (mg·L−1) | −80 °C | 438.37 | 395.33 | 388.57 | 347.48 | 543.55 | 273.54 | 318.30 | ||||
| −20 °C | 438.37 | 391.47 | 371.24 | 331.91 | 480.22 | 223.35 | 273.26 | 70.382 | <0.001 | 0.781 | 0.455 | |
| Average | 438.37 | 393.40 | 379.90 | 339.69 | 511.88 | 248.44 | 295.78 | |||||
| P1* | 0.031 | 0.001 | <0.001 | 0.002 | <0.001 | <0.001 | ||||||
| NH3-N (mg·dL−1) | −80 °C | 13.41 | 13.25 | 12.81 | 13.50 | 15.11 | 12.18 | 12.56 | ||||
| −20 °C | 13.41 | 13.20 | 12.35 | 12.61 | 13.74 | 12.61 | 11.60 | 2.166 | <0.001 | 0.885 | 0.637 | |
| Average | 13.41 | 13.22 | 12.58 | 13.05 | 14.43 | 12.40 | 12.03 | |||||
| P1* | 0.487 | 0.042 | 0.418 | 0.271 | 0.087 | 0.085 | ||||||
Note: 1 D0, D7, D14, D30, D60, D120, and D240 in preservation time indicate that rumen fluid was preserved for 0, 7, 14, 30, 60, 120, and 240 days, respectively. 2 P1* represents a pairwise comparison of frozen rumen fluid at a certain preservation time with fresh rumen fluid (D0). 3 SEM, standard errors of means. 4 The interaction effect in the p-value represents the interaction between preservation time and storage temperature.
Table 3.
Dynamic changes in volatile fatty acid concentrations (mmol/L) of rumen fluid with preservation time at two storage temperatures.
Table 3.
Dynamic changes in volatile fatty acid concentrations (mmol/L) of rumen fluid with preservation time at two storage temperatures.
| Item | Storage Temperature | Preservation Time 1 | SEM 3 | p-Value 4 | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| D0 | D7 | D14 | D30 | D60 | D120 | D240 | Time | Temperature | Interaction | |||
| Acetate | −80 °C | 40.70 | 40.15 | 39.39 | 42.93 | 46.42 | 44.62 | 40.54 | ||||
| −20 °C | 40.70 | 39.99 | 39.74 | 44.83 | 48.86 | 42.15 | 42.70 | 9.953 | <0.001 | 0.966 | 0.640 | |
| Average | 40.70 | 40.07 | 39.57 | 43.88 | 47.64 | 43.39 | 41.62 | |||||
| P1* 2 | 0.345 | 0.356 | 0.009 | 0.001 | 0.034 | 0.447 | ||||||
| Propionate | −80 °C | 9.84 | 9.39 | 9.13 | 9.90 | 11.24 | 11.07 | 10.23 | 3.225 | 0.022 | 0.964 | 0.752 |
| −20 °C | 9.84 | 9.41 | 9.30 | 10.24 | 12.12 | 10.68 | 10.69 | |||||
| Average | 9.84 | 9.40 | 9.21 | 10.07 | 11.68 | 10.88 | 10.46 | |||||
| P1* | 0.056 | 0.178 | 0.560 | 0.006 | 0.014 | 0.227 | ||||||
| Isobutyrate | −80 °C | 0.44 | 0.42 | 0.43 | 0.42 | 0.48 | 0.34 | 0.28 | <0.001 | 0.932 | 0.301 | |
| −20 °C | 0.44 | 0.42 | 0.43 | 0.53 | 0.47 | 0.32 | 0.28 | 0.068 | ||||
| Average | 0.44 | 0.42 | 0.43 | 0.47 | 0.47 | 0.33 | 0.28 | |||||
| P1* | 0.159 | 0.247 | 0.192 | 0.478 | <0.001 | <0.001 | ||||||
| Butyrate | −80 °C | 6.12 | 5.90 | 5.99 | 6.01 | 8.03 | 7.41 | 6.30 | 0.001 | 0.952 | 0.705 | |
| −20 °C | 6.12 | 5.82 | 5.82 | 6.66 | 8.34 | 7.20 | 6.98 | 1.974 | ||||
| Average | 6.12 | 5.86 | 5.91 | 6.34 | 8.18 | 7.30 | 6.64 | |||||
| P1* | 0.154 | 0.365 | 0.513 | 0.005 | 0.013 | 0.112 | ||||||
| Isovalerate | −80 °C | 0.76 | 0.74 | 0.76 | 0.73 | 0.60 | 0.56 | 0.44 | <0.001 | 0.907 | 0.107 | |
| −20 °C | 0.76 | 0.74 | 0.75 | 0.88 | 0.55 | 0.52 | 0.50 | 0.104 | ||||
| Average | 0.76 | 0.74 | 0.75 | 0.80 | 0.57 | 0.54 | 0.47 | |||||
| P1* | 0.321 | 0.590 | 0.295 | <0.001 | <0.001 | <0.001 | ||||||
| Valerate | −80 °C | 0.57 | 0.55 | 0.55 | 0.56 | 0.75 | 0.76 | 0.64 | <0.001 | 0.942 | 0.701 | |
| −20 °C | 0.57 | 0.55 | 0.55 | 0.61 | 0.78 | 0.73 | 0.69 | 0.124 | ||||
| Average | 0.57 | 0.55 | 0.55 | 0.58 | 0.76 | 0.75 | 0.67 | |||||
| P1* | 0.384 | 0.510 | 0.621 | <0.001 | <0.001 | <0.001 | ||||||
| Branched chain volatile fatty acids | −80 °C | 1.77 | 1.72 | 1.74 | 1.70 | 1.82 | 1.67 | 1.37 | <0.001 | 0.909 | 0.116 | |
| −20 °C | 1.77 | 1.71 | 1.73 | 2.02 | 1.80 | 1.56 | 1.47 | 0.237 | ||||
| Average | 1.77 | 1.71 | 1.73 | 1.86 | 1.81 | 1.61 | 1.42 | |||||
| P1* | 0.237 | 0.401 | 0.327 | 0.405 | 0.005 | <0.001 | ||||||
| Total volatile fatty acids | −80 °C | 58.44 | 57.17 | 56.26 | 60.54 | 67.52 | 64.77 | 58.43 | <0.001 | 0.962 | 0.741 | |
| −20 °C | 58.44 | 56.94 | 56.58 | 63.75 | 71.11 | 61.59 | 61.83 | 14.826 | ||||
| Average | 58.44 | 57.05 | 56.42 | 62.15 | 69.31 | 63.18 | 60.13 | |||||
| P1* | 0.170 | 0.288 | 0.054 | 0.001 | 0.020 | 0.374 | ||||||
Note: 1 D0, D7, D14, D30, D60, D120, and D240 in preservation time indicate that rumen fluid was preserved for 0, 7, 14, 30, 60, 120, and 240 days, respectively. 2 P1* represents a pairwise comparison of frozen rumen fluid at a certain preservation time with fresh rumen fluid (D0). 3 SEM, standard errors of means. 4 The interaction effect in the p-value represents the interaction between preservation time and storage temperature.
Table 4.
Dynamic changes in volatile fatty acid proportions (%) of rumen fluid with preservation time at two storage temperatures.
Table 4.
Dynamic changes in volatile fatty acid proportions (%) of rumen fluid with preservation time at two storage temperatures.
| Item | Storage Temperature | Preservation Time 1 | SEM 3 | p-Value 4 | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| D0 | D7 | D14 | D30 | D60 | D120 | D240 | Time | Temperature | Interaction | |||
| Acetate | −80 °C | 70.13 | 70.54 | 70.14 | 71.33 | 69.82 | 69.97 | 70.66 | ||||
| −20 °C | 70.13 | 70.46 | 70.42 | 70.44 | 69.86 | 69.52 | 70.30 | 1.083 | 0.227 | 0.889 | 0.669 | |
| Average | 70.13 | 70.50 | 70.28 | 70.88 | 69.84 | 69.74 | 70.48 | |||||
| P1* 2 | 0.204 | 0.653 | 0.041 | 0.518 | 0.378 | 0.457 | ||||||
| Propionate | −80 °C | 16.52 | 16.26 | 16.22 | 16.08 | 16.32 | 16.67 | 16.54 | 0.238 | 0.997 | 0.982 | |
| −20 °C | 16.52 | 16.21 | 16.21 | 16.06 | 16.34 | 16.71 | 16.61 | 1.496 | ||||
| Average | 16.52 | 16.24 | 16.21 | 16.07 | 16.33 | 16.69 | 16.57 | |||||
| P1* | 0.030 | 0.016 | 0.003 | 0.454 | 0.324 | 0.888 | ||||||
| Isobutyrate | −80 °C | 0.99 | 0.97 | 0.96 | 0.89 | 0.89 | 0.67 | 0.69 | <0.001 | 0.977 | 0.271 | |
| −20 °C | 0.99 | 0.96 | 0.97 | 1.05 | 0.87 | 0.68 | 0.61 | 0.182 | ||||
| Average | 0.99 | 0.97 | 0.97 | 0.97 | 0.88 | 0.68 | 0.65 | |||||
| P1* | 0.142 | 0.182 | 0.647 | 0.007 | <0.001 | <0.001 | ||||||
| Butyrate | −80 °C | 9.65 | 9.53 | 9.96 | 9.19 | 10.63 | 10.28 | 9.89 | 0.005 | 0.935 | 0.561 | |
| −20 °C | 9.65 | 9.65 | 9.67 | 9.73 | 10.67 | 10.66 | 10.15 | 1.285 | ||||
| Average | 9.65 | 9.59 | 9.81 | 9.46 | 10.65 | 10.47 | 10.02 | |||||
| P1* | 0.683 | 0.474 | 0.311 | 0.002 | 0.009 | 0.108 | ||||||
| Isovalerate | −80 °C | 1.68 | 1.65 | 1.67 | 1.53 | 1.10 | 1.12 | 1.00 | <0.001 | 0.939 | 0.420 | |
| −20 °C | 1.68 | 1.68 | 1.68 | 1.71 | 1.03 | 1.11 | 1.07 | 0.284 | ||||
| Average | 1.68 | 1.67 | 1.68 | 1.62 | 1.07 | 1.12 | 1.03 | |||||
| P1* | 0.296 | 0.907 | 0.095 | <0.001 | <0.001 | <0.001 | ||||||
| Valerate | −80 °C | 1.03 | 1.05 | 1.05 | 0.98 | 1.23 | 1.28 | 1.23 | <0.001 | 0.929 | 0.788 | |
| −20 °C | 1.03 | 1.04 | 1.05 | 1.01 | 1.22 | 1.31 | 1.26 | 0.093 | ||||
| Average | 1.03 | 1.05 | 1.05 | 0.99 | 1.22 | 1.30 | 1.24 | |||||
| P1* | 0.437 | 0.334 | 0.116 | 0.001 | <0.001 | 0.001 | ||||||
| Acetate/Propionate | −80 °C | 4.43 | 4.50 | 4.48 | 4.60 | 4.48 | 4.37 | 4.53 | 0.202 | 0.958 | 0.979 | |
| −20 °C | 4.43 | 4.51 | 4.50 | 4.53 | 4.46 | 4.33 | 4.43 | 0.393 | ||||
| Average | 4.43 | 4.50 | 4.49 | 4.56 | 4.47 | 4.35 | 4.48 | |||||
| P1* | 0.048 | 0.162 | 0.002 | 0.546 | 0.282 | 0.648 | ||||||
| Branched chain volatile fatty acids | −80 °C | 3.69 | 3.67 | 3.68 | 3.40 | 3.22 | 3.08 | 2.91 | <0.001 | 0.949 | 0.177 | |
| −20 °C | 3.69 | 3.68 | 3.70 | 3.78 | 3.12 | 3.10 | 2.94 | 0.546 | ||||
| Average | 3.69 | 3.68 | 3.69 | 3.59 | 3.17 | 3.09 | 2.92 | |||||
| P1* | 0.669 | 0.943 | 0.161 | <0.001 | <0.001 | <0.001 | ||||||
Note: 1 D0, D7, D14, D30, D60, D120, and D240 in preservation time indicate that rumen fluid was preserved for 0, 7, 14, 30, 60, 120, and 240 days, respectively. 2 P1* represents a pairwise comparison of frozen rumen fluid at a certain preservation time with fresh rumen fluid (D0). 3 SEM, standard errors of means. 4 The interaction effect in the p-value represents the interaction between preservation time and storage temperature.
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Fu, C.; Qu, M.; Ouyang, K.; Qiu, Q. Effects of Storage Time and Temperature on the Fermentation Characteristics of Rumen Fluid from a High-Forage Diet. Agriculture 2024, 14, 1481. https://doi.org/10.3390/agriculture14091481
AMA Style
Fu C, Qu M, Ouyang K, Qiu Q. Effects of Storage Time and Temperature on the Fermentation Characteristics of Rumen Fluid from a High-Forage Diet. Agriculture. 2024; 14(9):1481. https://doi.org/10.3390/agriculture14091481
Chicago/Turabian StyleFu, Chuanpei, Mingren Qu, Kehui Ouyang, and Qinghua Qiu. 2024. "Effects of Storage Time and Temperature on the Fermentation Characteristics of Rumen Fluid from a High-Forage Diet" Agriculture 14, no. 9: 1481. https://doi.org/10.3390/agriculture14091481
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