The Potential of Frozen Rumen Fluid with Dimethyl Sulfoxide And Glucose As Cryoprotectant on In Vitro Gas Production And Digestibility

. As the pressure from the animal welfare community increases, there is an urgent need to find the alternative rumen fluid (RF) source as an essential inoculum for in vitro digestibility studies. The objective of this study is to support the previous studies utilizing fresh RF from abattoir with and without cryoprotectants during the freezing process. A 2x3x3-factorial design was used to ascertain the effectiveness of dimethyl sulfoxide (DMSO) and glucose as cryoprotectants to frozen RF varying from 4, 11, and 18 days to measure the in vitro gas production (IVGP) and digestibility (IVD) of a complete feed consisted of elephant grass and commercial concentrate at 1:1 ratio. factor A being the preservation methods, factor B being the cryoprotectants, and factor C being the preservation time. IVGP shows that the use of DMSO and glucose as cryoprotectants (P1A, P1B, P2A, & P2B) only held back quality performance and made the non-cryoprotected frozen RF (P0A) is the most applicable method for preservation that lasts until 18 days. IVD however resulted in the most similar frozen RF treatment with gradual freezing that uses DMSO as cryoprotectant (P1B). Thus, making DMSO and glucose influenced nothing significant to the frozen RF. Maintaining the reduction of animal welfare while simultaneously keeping the data variation low is achievable and efficient.


Introduction
In vitro gas production (IVGP) and in vitro digestibility (IVD) are two widely used methods for evaluating the nutritional quality of complete feed formulations.IVGP measures the amount of gas produced during the fermentation of a feed sample by rumen microorganisms in vitro, while IVD determines the extent to which a feed sample is broken down and digested by rumen microorganisms in vitro.Rumen fluid (RF) as the medium for microorganism activities plays the role of rumen condition simulator.Obtaining it would be done in various way such as extraction by oral stomach tube, but prone to get contaminated to saliva.Another applicable way is to take it out from slaughtered cattle.It is more convenient reckoning the availability considered limited for live cattle extraction than the slaughtered ones that are procurable guaranteed.Freezing RF prevalent as preservation within 72 hours at 4°C; did not affecting gas production statistically, whilst decreasing at 96 hours [1].Frozen RF crystallizes that made microbe cells dehydrated decreasing the viability [2].
Sustaining the viability could be done by the addition of cryoprotectants such as glycerol or DMSO, which are proven to sustain microbe viability [3].Cryoprotectant could be not enough to preserve for more than 11 days, the gas produced decreased significantly [4].Pairing DMSO with glucose could help enhancing microbial activity [5], improving the accuracy of in vitro fermentation measurement [6], and also increasing ammonia concentration [7].
Combination of DMSO and glucose as cryoprotectants are expected to prevent dehydration on microbes from crystallization, which extend the frozen RF shelf life quality.

In Vitro On Ruminants
In vitro gas production (IVGP) is a technique used to estimate the fermentation capacity and nutritive value of feedstuffs and animal diets.In vitro gas production involves feed samples incubation with rumen fluid in a sealed container, and measuring the amount of gas produced as a result of microbial fermentation.The gas production is related to the degree of nutrient degradation and can be used to estimate the nutritive value of the feed or diet [8].Studies have demonstrated a strong correlation between IVGP and in vivo digestibility, particularly for forages [9].Compared to in vivo simulation, it is easier, cost efficient, time saving, control over experiments, and caliber to huge samples [8,10].
In vitro digestibility (IVD) is a laboratory technique used to measure the digestibility of feedstuffs and animal diets.It involves incubating a sample of feed or diet with digestive enzymes in a simulated rumen or intestinal environment, and measuring the amount of nutrients that are released during digestion.The degree of digestion is calculated by comparing the amount of nutrients in the original sample to the amount in the residual sample after digestion [9].
Rumen fluid (RF) that extracted from cattle, such as fistula one, oral stomach tube, or even feces, were considered as alternatives.However, they all have high risk, like for fistula cattle, prone to infections [12], oral stomach tube that is vulnerable to saliva and omasumabomasum fluid contamination [13], and low protozoa rate on feces [13].Based on all that, RF from slaughtered cattle is the best option considering the risk.Detailed and precise simulation is essential, meaning that the inoculum source should be the same genus as the cattle targeted to consume formulated feed.

Dimethyl Sulfoxide & Glucose as Cryoprotectants
Infamous by DMSO, it is a cryoprotective substance with high penetration rate [14].DMSO has high stability, high dielectric constant, alkali, solvation to salt (mainly anion), and tends to be an acceptor atom on hydrogen bonds (aprotic) [16].Binded DMSO and water in hydrogen resulting in a 30% stronger bond compared to the bond of two water molecules [17].In other words, DMSO could protect cells from biological damage in the cryopreservative process [18].
Based on its penetration method, DMSO is categorized as a cryoprotective substance that could permeate cell walls and cytoplasm membranes [19].Sub zero preservation without cryoprotectant, by default, is fatal.Cell freezing could be disadvantageous for results like crystallization which interfere with cell membranes to obtain a whole cell structurally after thawing, also increased concentration of dissolved solution at excessive liquid phase.The addition of cryoprotectant impacts on minimized cryobiology response, which is the shrinking of cells by osmotic pressure that inflates whenever the cryoprotectant penetrates to the cells [20, 21, 22, 23, 24, 25, and 26].
Glucose has been shown to be an effective cryoprotectant for a variety of biological samples, including animal embryos, stem cells, and tissues.Studies have demonstrated that the addition of glucose to cryopreservation solutions can improve survival rates and postthaw recovery of cells and tissues.Glucose has a wide range of applications in cryopreservation, including the preservation of animal embryos, stem cells, and tissues for medical and research purposes.It is also used in the cryopreservation of semen and oocytes for assisted reproductive technologies [27].Glucose also can interact with other cryoprotectants in cryopreservation solutions, affecting their efficacy and toxicity.Studies have shown that glucose can interact synergistically with other cryoprotectants, such as dimethyl sulfoxide (DMSO) and glycerol, to improve cryoprotection [5].

Material and Method
This research was conducted at Nutrition & Livestock Feed, Faculty of Animal Science, Universitas Brawijaya, from December 2022-March 2023.The objective was to determine by experiment that RF could still be useful after preservation for both IVGP & IVD.

Preparation
This experiment samples used complete feed that consisted of Elephant Grass (Pennisetum purpureum) from Sumbersekar Field Laboratory, Batu, & Mix Feed BAR A-18 concentrate by PT Berkat Agri Raya.The complete feed was portioned to be 1:1 ratio in each gram.
The RF used was extracted from Madura cattle at Gadang Slaughterhouse, Malang.It was extracted by digesta squeezing and pool scooping, which transferred into warm-watercontaining vacuum flask that disposed just before the RF.DMSO used was 1% and Glucose used was the anhydrous type by 0.8% modified the Pramita's [4].Immediately transferred into the laboratory, the RF are divided into two freezing methods, gradual (from rumen to room to refrigerator to chiller and to freezer temperature every 4 hours) and immediate, with each freezing method differentiated into 4, 11, and 18 days.The preservation executed in -22°C freezer, and when it is due simulation, thawing at 39°C waterbath is correct according to Fabro et al [1].

In Vitro
IVGP conducted according to Makkar et al. [28] for the chemicals, and used glass syringes & pistons that performed in a heated waterbath on 39°C for the technique.IVD on the other hand executed following Tilley & Terry [29] for the chemicals, and used glass fermenters that performed in incubators with the same temperature.

Variables
IVGP resulted in residue and gas produced, which will proceed into scoring such as total gas produced, potential gas produced, rate of gas produced, dry & organic matter for degradability, NH3 concentration, and microbial protein synthesis efficiency (MPSE).
IVD also resulted in residue that proceeded into scorings such as digestibility, dry matter, organic matter, ether extract, crude fiber, neutral detergent fiber, and acid detergent fiber.All of the variables are observed according to the AOAC [30].

Analysis
Analysis of the scores gathered was tabulated to ease up the analysis using variance (ANOVA) and will be proceeded with the Duncan test whenever resulting differences.The data are designed into randomized group design with 2x3x3 factorial pattern.Analyzed by ANOVA through Microsoft Office Excel with mathematics model from Steel & Torrie [31]: Analysis in the laboratory carried out results such as degradations, NH3 concentration, and microbial protein synthesis efficiency.Each variable resulted in lower score than the fresh RF but MPSE, by immediate freezing, all treatments overperformed the fresh RF.Dry matter degradability (DMD) on frozen RF came the closest with immediate freezing with no cryoprotectant at all by 46.9%.On organic matter degradability (DOM), the same scheme also resulted with no frozen RF scores matching the fresh RF.The closest was immediate freezing and no cryoprotectant treatment by 62.6%.NH3 concentration scored the most similar with DMSO only cryoprotectant by 6.2 and 6.9 mM.

Discussion
Degradation results indicated that freezing RF will underperform IVGP on DM or OM wise.By ANOVA, it showed that freezing RF and cryoprotect altered the DOM the most since all of the factors such as freezing, cryoprotectants, and preservation time resulted on more than 0.05 p-value.As the degradation results scored best on immediate freezing with no cryoprotectant, it can be assumed that cryoprotectant somehow did not preserve the rumen microorganisms.Principally, thawed frozen RF result should be 4-8 times lower than fresh RF [32,33], and also there could be DMSO toxicity increment when thawed since the experiment still did not result in a matching value according to fresh RF [34, 35, & 36].Visual interpretations were made on radar charts to illustrate the statistics of IVGP components.Fig. 1 depicts the non-cryoprotectant treatment on both immediate and gradual freezing.

Fig. 1. Visual Interpretations of Non-cryoprotected Treatments
The trend from immediate freezing shows that GP rate from frozen RF overperformed the fresh RF and peaking at 11 days (160%) after increased from 4 days (105%) then dipped at 18 days (125%) of preservation.However, the gradual freezing GP rate shows a different trend, peaking at 4 days (192.5%), then decreasing at 11 (110%) days and 18 days (92.5%).The different trend happened because of the cold shock which affected the metabolism process and made the immediate freezing score lower at 4 days of preservation [1].
Looking at the overperformed GP rate, it turned out total GP scored on both immediate and gradual freezing never even matched the fresh RF.Immediate freezing control treatment scored lower than the gradual freezing, which can be assumed that rumen microorganisms cannot survive the crystallization when frozen immediately from rumen temperature.Meanwhile the gradual freezing most likely survived by the temperature adjustment from rumen (39°C) to room (25°C) to refrigerator (4°C) to chiller (-4°C) then freezer (-21°C).The immediate freezing total GP trend was decreasing the longer the preservation, from 4 days (65%) to day 11 (60%) to 18 days (53%).However, on the gradual freezing there was no pattern, from 4 days (25.5%) to 11 days (76.6%) then to 18 days (72%).The similarities between both freezing methods is a verified phenomenon as the longer preservation occurs, the total GP will be lower [1].However, average total GP from non-cryoprotected frozen RF resulted under the optimum range between 76.73-149.90ml/500 mg DM [4].
The potential GP trends on both freezing treatments switched instead compared to the total GP.Immediate freezing potential GP did not have any pattern with 4 days (61.1%), 11 days (46.2%), and 18 days (50.3%) preservation time.On the other hand, gradual freezing potential GP shows that the longer the preservation time, the higher the potency with 4 days (23.1%), 11 days (68.1%), and 18 days (75.4%).Whenever cold shock affected the early preservation time, the potential GP resulted inverted since potential GP is not an undegraded feed, but potential to be instead [37].
Ruminal ammonia concentration is also altered by the freezing method; the longer it is preserved, the lower the NH3 concentration.Immediate freezing shows that the most similar concentration is on 4 days (86.8%) of preservation, followed by the decrease on 11 days (60.3%) and 18 days (49.2%).The gradual freezing resulted in the same trend with 4 days (75%), 11 days (70.6%), and 18 days (51.5%) of preservation.Decreased trends means that non-cryoprotectant frozen RF will lose microbial activity due to the ammoniac evaporation [38].Microbial protein synthesis however is higher than the NH3 concentration when compared to the fresh rumen like it is contrary.Immediate freezing has a hill-like trend that it increased on 11 days (127.7%) after 4 days (75.7%) and then decreased at 18 days (105.2%).Meanwhile the gradual freezing has a valley-like trend which peaked at 4 days (118.2%) and then dipped at 11 days (44.7%) and then rose up again at 18 days (103%).It was mentioned that MPSE scored higher than NH3 concentration which the ruminal ammonia themselves averaging lower than the fresh, and only two treatments from immediate and gradual freezing at 4 days (7.1 & 6.1 mM) reached 6 mM as the minimum requirement for MPSE [37], means there was an inefficient N usage which made the MPSE overperformed the fresh RF [39].
It could be summarized that frozen RF without cryoprotectant needs to be adjusted temperature wise to keep the rumen microorganisms' viability.Besides freezing without cryoprotectants did not preserve rumen microorganisms, it altered the GP rate and MPSE to be contrasting the total & potential GP, and the NH3 concentration.

Fig. 2. Visual Interpretations of DMSO cryoprotectant Treatments
The Fig. 2 presented that GP rate trend is still the same while already cryoprotected with DMSO.Both freezing methods overperformed the fresh RF.Immediate freezing has the same trend by peaking in the middle with 4 days (147.5%),11 days (190%), and 18 days (102.5%) as the preservation time.On the other hand, gradual freezing summoned a new trend by decreasing then constant with 4 days (177.5%),11 days (117.5%), and 18 days (117.5%)preservation time.Cold shock by immediate freezing resulted in decreasing score while gradual freezing served a GP rate consistency [1].
Compared to the non-cryoprotected frozen RF, with DMSO the GP resulted in more similarity to the fresh RF GP.Total GP between immediate and gradual freezing has the same trend with increment.Immediate freezing has the valley with 4 days (70.4%), 11 days (61.8%), and 18 days (87.8%) preservation time.Meanwhile the gradual freezing always increased by 4 days (56.5%), 11 days (60.7%), and 18 days (87.1%) preservation time.Averaging higher GP than non-cryoprotected frozen RF, the longest preservation time resulted in the highest instead while keeping the optimum range of total GP [1,4].However, the higher total GP compared to the control could be generated by the cryoprotectants, not the ruminal microorganisms.
The potential GP having a different trend compared to the total GP.Immediate freezing resulted in a valley-like trend with 4 days (54.4%), 11 days (48.7%), and 18 days (91.1%) as the preservation time.Gradual freezing tends to have an increment as the trend with 4 days (45.9%), 11 days (54.2%), and 18 days (84.8%) long preservation time.Difference between how frozen RF prepared and composed will affect the GP, in this case potential GP resulted more similarly to the fresh RF compared to the non-cryoprotected [40].
DMSO as cryoprotectant preserved the ruminal ammonia concentration longer than without any cryoprotectant.Immediate freezing with DMSO resulted in 80.9% similarity to fresh RF on both 4 and 11 days of preservation, while the score decreased to 65.4% at 18 days.Through gradual freezing, the NH3 concentration overperformed but ended with the same decreased trend.Started high on 4 days (117.6%), to 11 days (92.6%), and 18 days (42.6%) of preservation time.DMSO preserved better than non-cryoprotected since the decreased concentration below 70% similarity only occurred on the longest preservation and had lost its microbial activity due to the evaporated ammonia [1,38].
The single cryoprotectant overperformed more stable on MPSE when compared to fresh RF.On immediate freezing, the trend is always increasing scores with 4 days (118.9%),11 days (127.1%), and 18 days (128.1%) of preservation time.Gradual freezing had the decrease at the end with 4 days (79.8%), 11 days (114%), and 18 days (93.9%) preservation times.Constant similarities to the fresh RF except for the gradual freezing at 4 days means that DMSO preserved the fresh RF quality.On the flipside, the overperformance indicated that lower rumen ammonia-N concentration did not limit the MPS efficiency [41].
Wrapped trends interpret that preserving frozen RF best using DMSO as the cryoprotectant with immediate freezing since only NH3 concentration scores higher on the other freezing method, while the rest variables like total, potential, & rate of GP, and MPSE scored the same with fresh RF.As DMSO works on membrane cell level by inhibiting cell dehydration along the preservation [42], it really resulted in better scores than noncryoprotected frozen RF, which made the cryoprotectant preserve the fresh RF quality.

Fig. 3. Visual Interpretations of DMO & Glucose cryoprotected Treatments
The third treatment is cryopreserving frozen RF using DMSO & glucose as pairing.The GP rate still overperformed the fresh RF but not as high as using DMSO only as the cryoprotectant.Immediate freezing generated GP higher by hill-like trend with 4 days (132.5%),11 days (180%), and 18 days (97.5%) preservation times.Meanwhile the gradual freezing GP rate resulted in a valley-like trend with also 4 days (150%), 11 days (107.5%), and 18 days (122.5%)preservation times.Cryoprotectant pairings resulted in a more similar result to the fresh RF even though still overperformed.
The pairing of DMSO & glucose resulted best on total GP on both freezing method and having similar trends with increment.Immediate freezing preserved the fresh RF trait best, since it scored higher than the other method, with 4 days (73.4%), 11 days (73.3%), and 18 days (85.9%) preservation times.The gradual freezing method scored an increasing trend with 4 days (66.1%), 11 days (71.5%), and 18 days (87%) of preservation time.Stable trend shown on the immediate freezing method means that the synergic effect of pairing actually happened [15].
Potential GP resulted best in cryoprotectants pairing than singular use.Immediate freezing preserved better scores by valley-like trend with 4 days (62.9%), 11 days (59.6%), and 18 days (75.4%) preservation times.On the other hand, the gradual freezing has an increasing trend with 4 days (58.1%), 11 days (65.4%), and 18 days (79.6%) of preservation time as indicators.Averaged higher than the gradual method, immediate freezing could preserve higher protein since the gradual method might have microbial activity loss from the interval of slow temperature change [43].
However, the NH3 concentration scored the lowest with cryoprotectants pairing.Immediate freezing turned out to be the most unlikely preserved fresh RF quality with 4 days (50%), 11 days (54.4%), and 18 days (72.1%) of preservation time that increased late.The gradual freezing resulted better instead with the same 4 days (66.2%), 11 days (82.4%), and 18 days (42.6%) preservation times.Since the assumed protein was preserved higher on the immediate freezing by kept quality of microbial activity, the NH3 concentration resulted lower [44].
Contrary to the NH3 concentration which were very low to meet the minimum MPSE requirement, the MPSE scores resulted high and overperformed with decreasing trends.Immediate freezing method averaged highest with 4 days (130.8%),11 days (142.7%), and 18 days (125.1%)preservation times.On the other hand, gradual freezing averaged second best with 4 days (135.4%),11 days (128.3%), and 18 days (114.5%) of preservation time.As MPSE average highest on DMSO & glucose pairing, the phenomenon verified as decreased fermentation happened to lyophilization RF that thawed without sugar or growth promoters.In this case, the frozen RF had both combined [45].
The same tendency spotted in the pairings of DMSO & glucose that immediate freezing method resulted the most similar to fresh RF but NH3 concentration.Derived from fungi preserved fermentation using 10% DMSO & 8% glucose [46], and not so well performed frozen RF with 5% DMSO (Pramita, 2022), the use of reduced doses to 1% DMSO & 0.8% glucose through immediate freezing made it as the second most similar performance to the fresh RF.

Fig. 4. Visual Comparisons of Freezing Methods
More detailed analysis needs to be carried out.After proceeded into Steel & Torrie model of ANOVA and continued to Duncan test whenever differences conducted, it can be concluded that there were no differences between frozen RF treatments, except for degradations, which significantly affected from variations of cryoprotectant usage and influenced by freezing methods as presented in the table 3.However, when compared to the fresh RF (table 4), freezing actually altered every variable observed significantly but potential GP and OM degradation.Fig. 4 shows that immediate freezing results are more similar to the fresh RF.

Fig. 5. Total Gas Produced On 4 Days Preservation Graphic
Fig. 5 shows the total GP graphic comparison between fresh RF and 4 days of preservation for frozen RF.Different freezing methods resulted in different behaviour that immediate freezing produced the highest at the 24 th hour, all of the cryoprotectant treatments, with no cryoprotectant (P0A), DMSO only (P1A), and pairings of DMSO & glucose (P2A).Meanwhile the gradual method peaked at 8 th hour with P0B, P1B, and P2B uniform in trend.However, the targeted performance according to the fresh RF, highest GP generated in the 48 th hour.Fig. 6 shows the inverted behaviour between freezing method variations.Immediate freezing with its P0A, P1A, & P2A at 11 days of preservation produced the most at 8 th hour.Meanwhile gradual freezing with P0B, P1B, & P2B peaking the GP at 24 th hour.Nevertheless, the fresh RF still accumulated higher total GP and behaved differently.Fig. 7. Total Gas Produced On 18 Days Preservation Graphic Fig. 7 shows another pattern of behaviour from frozen RF total GP.Only P0A from immediate freezing generated the most GP at 8 th hour, whilst the rest from both freezing methods such as P1A, P2A, P0B, P1B, & P2B were all peaking at the 24 th hour.Compared to the fresh RF as the desired result, total GP wise, 18 days long preservation time accumulated the most similar as the data can be seen on table 1.

Fig. 8. Total Gas Produced Graphic
Fig. 8 shows that the peak of GP happened on the 8 th hour for both freezing methods.While the highest GP generated by fresh RF as the target occurred at 48 th hour, observing the trend from the graphic, fresh RF second highest GP also peaked at the 8 th hour.3 summarized the factor A & B namely freezing and cryoprotection methods.Percentages indicating performance similarities by frozen RF compared to the fresh RF.Pvalues show different significance between frozen RF, with only degradations, both DM & OM, having alternation by freezing method (F), cryoprotectants (C), and the interactions (F><C).
The most similar results compared to fresh RF was non-cryoprotected RF that froze immediately.However, the total & potential GP is considered low.The most applicable cryoprotectant that scored similarly would be pairings of DMSO & glucose with immediate freezing, even though the MPSE performance far surpassed the fresh RF.Since there were two most performing frozen RF treatments that were similar to the fresh RF, analysis on difference significance from fresh and frozen RF was conducted.Generated through SPSS by IBM, independent T-test applied to 18 different frozen RF treatments and one treatment with fresh RF.It was stated that potential GP and OM degradability are not significant, yet the total GP and DM degradability significance only exceeded a bit from the 0.05 as the indicator.Hence why it can be concluded that total & potential GP and also DM & OM degradability do not matter preservation wise.Means that the use of no cryoprotectant is the most applicable preservation method that lasts to 18 days and still performing the most similar to the fresh RF, since GP differences could be caused by McDoughal's solution that produced extras [28,4].Residual nutritive content that is carried out by proximate analysis (except protein) shows that digestive ability of frozen RF is still below the fresh RF, since the preservations resulted higher.Dry matter of the fresh RF scored at 42.7%, while for the most similar from the frozen RF came from gradual freezing with DMSO by 48%.Organic matter content from residue maximized by fresh RF at 37.8% and scored the most similar with the same gradual freezing that used DMSO as the cryoprotectant.The ether extract showed a different trend from the cryoprotectant aspect while still maintaining the gradual freezing corridor, 5.9% EE resulted from the same feed sample while the targeted score is higher at 6.8%.Crude fiber desired at 27.8% as the maximum value, while the most similar scored by gradual freezing without any cryoprotectant at 28.4%.As the fiber might not be enough when only crude, detailed fiber breakdowns are conducted through analysis of neutral and acid detergent fiber.NDF functioned as the identifier of less digestible fiber like hemicellulose, cellulose, and lignin.While ADF functioned as the mostly digestible fiber identifier such as starch and sugar.
NDF as the less digestible became more unlikely to be digested by using the freezing method.The targeted result scored by fresh RF at 52.6% is digested maximum since the microbial activity is still unaltered.Meanwhile the most similar for frozen RF was without any cryoprotection and froze immediately by 56.1%.
ADF showed a different trend that every digested fiber that ran through frozen RF resulted lower.The fresh RF being the highest with 47.4% followed by the most similar with gradual freezing that used no cryoprotection by 44.3%.However, the most digested ADF was the gradual freezing method with DMSO at 34.5%.Total digestibility presented by DM & OM digestibility.Frozen RF treatments resulted in lower digestibility, which can be assumed that preservation lost some of the microbial activity.Digested dry matter targeted at 59.5% with fresh RF, while the most similar result from frozen RF carried out by the treatment of gradual freezing with DMSO at 49.8% as the best digestibility through preservation.Organic matter digestibility is desired at 68.9% from the fresh RF followed by the most similar from gradual freezing with DMSO at 57.8%.

Discussions
Most of the similar performance from IVD compared to the fresh RF resulted from gradual freezing with DMSO as cryoprotectant.The only deviants were EE and CF with the most similarities on gradual freezing without any cryoprotection.While principally the higher the CF it would be harder to digest [47], this experimental research only used the same feed sample as forage and concentrate like elephant grass and commercial mixfeed.However, the fermentative results lower on frozen RF and can be assumed that the microbial activity decreased due to some dead microbes in it or could be caused by an unstable thawing method too [48,34].Table 9 shows how significant the frozen RF treatments altered the variables.Ran through SPSS by IBM using independent T-test analysis, it generated summaries that DM, content or digestibility, did not alter at all.EE being lower than the fresh RF had no significance compared to the frozen RF, which means scored lower solely just because the microbial activity loss.NDF applied the same principle due to the preservation.

Conclusion
The use of cryoprotectant in the DMSO form turns out to effectively preserve the quality of fresh RF to proceed into in vitro analysis.However, IVGP needs to be studied and experimented further since the most similar result came from cryoprotectants pairing that froze immediately, but was doubted because of its GP results.Degradation wise, the result was found most similar to gradual freezing with DMSO only.It is the same pattern as the IVD result.
Running IVGP with frozen RF at this point is still recommended to be using no cryoprotectant at all.While for IVD, it is recommended to have gradual freezing with DMSO as preservation method.

Table 1 .
Gas Production Analysis Results from IVGP

Table 3 .
Summary of IVGP Experiment Results

Table 4 .
Results of Independent T-Test Between Fresh and Frozen RF Through IVGP

Table 5 .
Proximate Analysis Results from IVD

Table 6 .
van Soest Fiber Analysis Results from IVD

Table 7 .
Digestibility Analysis Results from IVD

Table 8 .
Summary of IVD Experiment Results

Table 9 .
Results of Independent T-Test Between Fresh and Frozen RF Through IVDThe most similar value scored as fresh RF was gradual freezing with DMSO as the cryoprotectant.It can be applied into long in vitro experiments as the preservation method that lasts until 18 days.BIO Web of Conferences 88, 00009 (2024) ICIAS 2023 https://doi.org/10.1051/bioconf/20248800009