Effect of Dietary Non-Fiber Carbohydrate Sources and Sulfur Supplementation on in vitro Ruminal Fermentation and Digestibility of the Dairy Ration

Iranian Journal of Applied Animal Science

دوره 13، شماره 2، شهریور 2023، صفحه 231-240   

نوع مقاله: Research Articles

نویسندگان

A. Rosmalia؛ I.G. Permana^* ؛ D. Despal؛ T. Toharmat؛ F.R. Pambudi؛ S.I.Z. Arif

Department of Animal Nutrition and Feed Technology, Faculty of Animal Science, IPB University, Jl. Agatis, Kampus IPB Dramaga, Bogor, West Java, Indonesia

چکیده

Synchronization of rumen degradable protein (RDP), non-fiber carbohydrate (NFC), and sulfur availability are needed for optimum microbial protein synthesis (MPS), rumen fermentation activity, and feed digestibility. Cassava and corn are rich in NFC content and have different carbohydrate characteristics. Most of the tropical feedstuff is deficient in sulfur, thus it needs to be supplemented in the ration. This study aimed to compare the effect of corn and cassava as NFC sources and sulfur supplementation on fermentability and digestibility using in vitro study. The experiment used a 2 × 3 factorial randomized block design with four different dairy cattle rumen liquor as replications (block). The first factor was NFC sources (corn and cassava), and the second factor was the level of sulfur supplementation (0%, 0.1%, 0.2%). Parameters observed include fermentability (rumen pH, NH₃ concentration, total volatile fatty acids (VFA), molar proportion of VFA, MPS, rumen bacteria, and protozoa population) and in vitro digestibility (dry matter (IVDMD) and organic matter (IVOMD)). Data were tested using ANOVA followed by the Duncan test. The result showed an interaction between rumen pH (P<0.05). The NH₃ concentration was low in cassava treatment, while total VFA did not have a significant effect. Corn treatment produced a higher iso-butyrate and iso-valerate than cassava treatment (P<0.05). The rumen microbes, MPS, IVDMD, and IVDMD, did not differ among the treatments. Cassava could replace corn as an NFC source for tropical dairy ration along with providing RDP in balance ratio and sulfur supplementation.

کلیدواژه‌ها

in vitro digestibility؛ non-fiber carbohydrate؛ rumen degradable protein؛ sulfur

اصل مقاله

INTRODUCTION

The global demand for animal foods is growing along with rising population growth and economic prosperity (Henchion et al. 2021). This is a challenge to providing human food and preserving human health by increasing livestock production. Milk and its product are nutrient-rich foods that largely contributes to human nutrition (Smith et al. 2022). Nowadays, the dairy production system has focused on their sustainability by considering animal welfare, feed efficiency, and environmental impact. Improving feed efficiency can be achieved by improving feed quality, identifying alternative feed ingredients, better explaining nutrient requirements, improving the efficiency of ruminal fermentation, and increasing dietary digestibility (Eastridge, 2006). Ruminal fermentation and digestibility is the main factor to supply nutrients for dairy cows, indeed for their maintenance and as a precursor of milk synthesis. Some factors that alter fermentation in the rumen and nutrient digestibility are the type of diet, forage-to-concentrate ratio, physical form, chewing activity, particle size (Li et al. 2020), rate of passage, carbohydrate source (Srakaew et al. 2021), dietary protein (Firkins et al. 2007), and rumen microbial population (Castillo-González et al. 2014). Determining the type of diet, mainly carbohydrates and protein, is necessary to meet microbial growth for improving ruminal fermentation and digestibility. Many previous studies have been investigated regarding arranged feed protein by supplying rumen degradable protein to undegradable protein ratio (RDPR) (Kaufman et al. 2018; Rosmalia et al. 2021; Sahroni et al. 2021), readily available carbohydrates reflected by non-fiber carbohydrate (NFC) (Mertens 1997; Hall et al. 2010; Villalba et al. 2021), and energy-protein synchronization (Sun et al. 2019; Rosmalia et al. 2022a). Corn and cassava are the energy source feed ingredients that provide a fairly high NFC (Kanjanapruthipong et al. 2001). Corn is widely used in dairy rations and is slow degradation (Herrera-Saldana et al. 1990). Corn has a starch content of about 80% (Hall et al. 2010). Cassava meal is a feed ingredient derived from cassava (Manihot esculenta) and is commonly used as an energy source for livestock in tropical areas (Wanapat et al. 2018). As a fermentable energy source, cassava has cell walls rich in pectin (Staack et al. 2019). The pectin content in the cassava cell wall is 35.7% (Nurdjanah and Elfira, 2009). The degradation rate of cassava is higher than that of corn (Wanapat et al. 2018). The results of previous studies stated that the type of NFC that interacts with RDP affects rumen fermentation and microbial growth (Zhao et al. 2020). Besides considering the synchronization between RDP and NFC sources, sulfur supplementation in dairy rations is necessary to optimize the production of microbial protein synthesis (Rebelo et al. 2019). Sulfur plays a role in sulfur amino acid and vitamin synthesis (Miller, 1979). However, the sulfur content in the tropical dairy ration is deficient due to the use of agricultural by-products (Promkot and Wanapat, 2009). A sulfur deficiency was associated with reduced milk yield and milk fever (Pieper et al. 2016). Thus, it needs to add to the dairy ration. Investigating the association of NFC sources and sulfur supplementation in the tropical dairy ration has not been studied. Therefore, the objective of this study was to compare the effect of corn and cassava as an NFC source and sulfur supplementation on the fermentability and digestibility of dairy ration in tropical areas.

 

MATERIALS AND METHODS

The animal used was housed and cared for according to the Animal Ethics Committee, IPB University guidelines. This study was also proved within contract No. 047/KEH/SKE/XI/2021.

 

Experimental diet

The experimental diet used in this study was a 2 × 3 factorial randomized block design. The first factor was NFC sources (corn and cassava), and the second was sulfur supplementation (0%, 0.1%, 0.2%). The formulated diet is based on the NRC dairy requirement within 14% CP, 60:40 RDP to rumen undegradable protein (RUP) ratio, 35% NFC level, and set 50:50 for forage to concentrate ratio (dry matter basis). Feedstuff consisted of elephant grass, corn, rice bran, pollard, molasses, copra meal, palm kernel meal, soybean meal, corn gluten meal (CGM), and distillers dried grains with solubles (DDGS). The mineral sources were dicalcium phosphate and sodium sulfate (Na₂SO₄) as sources of sulfur. Feedstuff was finely ground and mixed through the feed formulation in Table 1. The nutrient content of the treatment diet is shown in Table 2 and it was analyzed according to the AOAC method (AOAC, 2005).

 

In vitro study and sample analysis

The treatment diet was carried out by in vitro two-stage procedures (Tilley and Terry, 1963), similar to Despal et al. (2022). The rumen fluid was obtained from rumen-fistulated dairy steers (510 kg of body weight) and collected before morning feeding. The McDougall solution was used as a buffer solution, which was made by diluting 9.8 g NaHCO₃, 4.65 g Na₂HPO₄.2H₂O, 0.57 KCl, 0.47 g NaCl, 0.12 g MgSO₄.7H₂O, and 0.04 g CaCl₂ into 1 L distilled water. A 0.5 g sample was weighed and placed into a fermentor tube. For ruminal fermentation (stage 1), the samples were incubated in 10 mL rumen fluid and 40 mL McDougall buffer solution for four h at 39 ˚C in a shaker water bath under anaerobic conditions. After four h of ruminal incubation, 0.5 mL, 1 mL, and 20 mL of samples were taken using a syringe for rumen bacteria and protozoa count, and microbial protein synthesis, respectively. The pH value was measured using a pH meter (Hanna Instrument, HI98191). For the molar proportion of individual volatile fatty acids (VFA), 5 mL of sample was collected, and added one drop of H₂SO₄ 98% were then stored at -20 ˚C for further analysis. The fermentation process was ended up with adding two drops of HgCl₂ solution. The supernatant was collected by centrifugation at 3500 rpm for 15 min and then stored in a freezer to analyze of NH₃ concentration, total VFA concentration, and VFA profiles. The in vitro digestibility measurements, including dry matter and organic matter (IVDMD and IVOMD), were conducted through the fermentation process (stage 1) and enzymatic process (stage 2). In the first stage, the sample was incubated with rumen and McDougall buffer solution for 48 h at 39 ˚C. Afterward, two drops of HgCl₂ solution were added to stop the microbial activity.

 

Table 1 Feed formulation of the treatment diets

CGM: corn gluten meal; DDGS: distillers dried grains with solubles and DCP: dicalcium phosphate.

 

Table 2 Chemical composition of the treatment diets

NFE: nitrogen free extract and TDN: total digestible nutrient.

TDN= 2.79 + 1.17 CP + 1.74 EE – 0.295 CF + 0.81 NFE according to Sutardi (1980) in (Indah et al. 2020).

 

The sample was separated into supernatant and residue by centrifugation at 3500 rpm for 15 min. In the second stage, the supernatant was removed, then the sample residue was incorporated with 50 mL of pepsin-HCl solution. The pepsin solution was prepared by diluting 2 g pepsin (10000 NF U mg^-1, from the bovine origin, HIMedia) in 17.8 mL of 37% HCl and 1000 mL of distilled water. The tube was incubated aerobically for 48 h at 39 ˚C in a shaker waterbath. Two drops of HgCl₂ were added to halt enzymatic activity. Then, samples were filtered (Whatman No. 41 ashless filter paper), and the filter paper and residue were dried in an oven at 100 ˚C for 24 h and finally weighed to determine the residue's dry matter. Next, the dry residue was incinerated in an oven furnace at 650 ˚C for four h and subsequently weighed to determine the ash of the residue. The IVDMD was calculated by diminishing the dry matter of residue (corrected for the blank) from the dry matter of the sample. In comparison, the IVOMD was calculated by subtracting 100% from the ash of residue (corrected for the blank) and the initial sample. The frozen sample of NH₃ and total VFA concentration were thawed at room temperature (25 ˚C). The NH₃ concentration was measured using the Conway microdiffusion method, while the total VFA concentration was conducted through the steam distillation method referred to (Permana et al. 2022). Gas chromatography was run to obtain the molar proportion of individual VFA (Yulistiani et al. 2017). Microbial protein synthesis (MPS) was determined according to Makkar and Lowry's protein method (Makkar et al. 1982). The Ogimoto and Imai method calculates rumen bacteria and protozoa population (Rosmalia et al. 2022b).

 

Statistical analysis

The study used a 2 × 3 factorial randomized block design with four different dairy cattle rumen liquor as replications. Data were analyzed using the SAS OnDemand for Academic (SAS, 2004) for variance analysis. Effects of the factors were declared significant at P ≤ 0.05 and further tested by Duncan's Multiple Range Test.

 

RESULTS AND DISCUSSION

Ruminal fermentation

The fermentability of the treatment diet including is shown in Table 3. There was a significant interaction between NFC sources and the level of sulfur supplementation on rumen pH. A high rumen pH value resulted from cassava treatment with sulfur supplementation and corn treatment with 0.2% sulfur supplementation. NFC sources significantly influence rumen pH. Corn treatment decreased ruminal pH.

 

Table 3 The fermentability of the treatment diet including rumen pH, NH₃ concentration, and total VFA concentration

VFA: volatile fatty acids.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

 

There was no interaction between the two factors, but the main factor, NFC sources, showed a significant difference in the NH₃ concentration. The NH₃ concentration in the corn treatment was higher than in the cassava treatment. Total VFA concentration was not significantly influenced by NFC source, sulfur supplementation, or their interaction. The NH₃ and total VFA concentrations produced in this study were in the normal ranges, ranging from 5.13-6.92 mM and 105.05-152.14 mM, respectively (McDonald et al. 2010; Dieho et al. 2016). The VFA molar proportions of the treatment diets are presented in Table 4. Based on statistical analysis, the main factors and their interaction did not affect the molar proportion of acetate, propionate, n-butyrate, n-valerate, and acetate to propionate ratio. Iso-butyrate and iso-valerate were significantly affected by NFC sources. Corn treatment produced a higher iso-butyrate and iso-valerate than cassava treatment.

 

Rumen microbes and microbial protein synthesis

The rumen microbes population indicates whether the rumen degradation was doing well. Ruminal microbial population (bacteria and protozoa) and MPS are shown in Table 5. There was no interaction between NFC sources and sulfur supplementation and the effects of the main factors on ruminal bacteria, protozoa population, and MPS. The number of rumen bacteria found in this study was slightly higher (10¹¹-10¹² CFU mL^-1) than the normal range (10¹⁰-10¹¹ CFU mL^-1) (Bainbridge et al. 2018), while the protozoa population was in the normal condition (6.48-6.62 log cell mL^-1) (Matthews et al. 2019). The means of MPS ranged from 5.36-6.77 mg 10 mL^-1.

 

In vitro dry matter and organic matter digestibility (IVDMD and IVOMD)

The IVDMD and IVOMD of the treatment diet are presented in Table 6. Either the main factor and their interaction (NFC sources and sulfur supplementation) were not significantly affected by IVDMD and IVOMD. The mean of IVDMD and IVOMD in this study ranged from 68.80-69.76% and 72.27-73.15%, respectively. The treatment diets changed rumen pH. Therefore, the ruminal pH in this study was in the normal condition (6.00-7.00) to support microbial activity and growth (McDonald et al. 2010). The high pH in 0.2% sulfur supplementation with NFC sources corn and cassava with all levels of sulfur (P<0.05). In contrast, a previous study found that a high sulfur diet can lower rumen pH by increasing H₂S concentration in the rumen (Felix and Loerch, 2011). The high rumen pH in this study might be due to increasing effective NDF in the ration, which leads to decreasing H₂S and H⁺ (Drewnoski et al. 2014), and the treatment diets have a good buffering capacity (Wu et al. 2015). Rumen pH was influenced by diet properties, saliva secretion, osmotic pressure, volatile fatty acids, rumen water flow, and buffering power of feed (Zheng et al. 2020). The NH₃ concentration produced in this study was sufficient for microbial protein synthesis. The minimum NH₃ concentration is 2.94 mM, while the optimum to support microbial growth is 5.00-17.65 mM (McDonald et al. 2010). The cassava treatment had a lower NH₃ concentration than the corn treatment. A previous study also reported that the cassava diet produced low NH₃ compared to the corn diet (Putridinanti et al. 2019). Zheng et al. (2020) revealed that the inclusion of cassava in the diet reduced the NH₃ concentration related to high carbohydrate content in cassava treatment. The high availability of carbohydrates stimulated the rumen microbes to absorb the nitrogen, then decreased in the NH₃ concentration (Henning et al. 1991). The total VFA produced in this study was higher than Dieho et al. (2016), 105.05-152.04 mM and 100.96-121.20 mM, respectively.

 

Table 4 VFA molar proportion of the treatment diets

 

C2/C3: acetate to propionate ratio and VFA: volatile fatty acids.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

 

Table 5 Ruminal microbial population and microbial protein synthesis

 

This indicates that the treatment diet based on corn and cassava as NFC sources was highly fermentable and supplied sufficient energy for rumen microbes and the host (Wang et al. 2020). The high production of total VFA also indicated high organic matter that was degraded easily by rumen microbes (Joo et al. 2005). Although corn and cassava had different characteristics of carbohydrates, the total VFA in this study did not differ among the treatments diet. Different types of carbohydrates could change the VFA molar proportion (Bannink et al. 2006). Many factors could change the total VFA concentration, such as substrate availability, rumen fermentation rate, feed consumption, VFA absorption, and liquid or solid passage (Hall et al. 2015).

 

Table 6 In vitro dry matter and organic matter digestibility of the treatment diets

IVDMD: in vitro dry matter digestibility and IVOMD: in vitro organic matter digestibility.

 

Propionate is one of the precursors of lactose formation for dairy cows and a substrate for hepatic gluconeogenesis (Habel et al. 2022). Propionate concentration was higher in the rations with low forages (Sutton et al. 2003). The addition of readily degradable carbohydrates in the ration will increase propionate production (Chanjula et al. 2007). Based on the results of this study, sulfur treatment and NFC type did not significantly effect on the molar proportion of propionate. This aligns with a previous study that the propionate concentration between corn-based rations and cassava had no significant effect (Chanjula et al. 2007). This is due to the similar level of NFC in corn and cassava treatment, so the propionate production had no difference. The results of this study are also in line with Zhao et al. (2020), which state that Na₂SO₄ supplementation did not affect the molar proportion of propionate. Fibrobacter bacteria that produce succinate are not affected by Na₂SO₄ supplementation, so propionate production is not disturbed because succinate can be converted into propionate by succinate-to-propionate bacteria. VFA branched chains, such as iso-butyrate and iso-valerate, are products of oxidative deamination and decarboxylation of amino acids in the form of isoleucine, valine, and leucine (Rosmalia et al. 2022c). Corn treatment produced high iso-valerate and iso-butyrate compared to cassava treatment. This is in line with Zheng et al. (2020), who reported that the production of iso-valerate and iso-butyrate tends to decrease due to substituting of corn with cassava meal. The protein content in corn treatment was higher than in cassava treatment resulting in a high molar proportion of iso-valerate and iso-butyrate in corn treatment. Based on this research, there is no effect on the production of n-butyrate and n-valerate. Zheng et al. (2020) also reported that corn produced higher valerate production than cassava treatment, with no effect on butyrate. The sulfur supplementation with Na₂SO₄ did not affect the concentration of butyrate. The study’s result differed from Zhao et al. (2020), which reported that Na₂SO₄ supplementation increases butyrate production due to increased growth of Butyrivibrio fibrisolvens and Pseudobutyrivibrio spp. Bacteria. Acetate is the main precursor for dairy livestock to synthesize of milk fat (Urrutia and Harvatine, 2017). In this study, there was no interaction between sulfur supplementation levels and NFC types, and no significant effect of both treatments on the molar proportion of acetate and acetate to propionate ratio. This might be due to the similar NFC levels in the ration. The molar proportion of acetate will increase along with the increase in NFC levels (Rosmalia et al. 2022a). Chanjula et al. (2007) also reported that acetate concentrations did not significantly effect between corn-based rations and cassava meal. The ratio between acetate and propionate did not differ markedly as cassava meal supplementation increased into corn-based rations. In contrast, Zhao et al. (2020) revealed that Na₂SO₄ supplementation might increase acetate production due to increased Ruminococcaceae bacteria. Rumen bacteria are the most abundant microbes in the rumen and contribute to enzymatic activities (amylase, lipase, protease, cellulase, and xylanase). Protozoa play roles in fermentation, especially in the rumen pH, and detoxification of mycotoxin. They are also closely associated with methanogenic archaea (Elghandour et al. 2019). The main factor of rumen microbial growth and activity include pH, temperature, buffer capacity, osmotic pressure, dry matter content, and oxidation-reduction potential. The rumen bacteria population found in this study was slightly higher than the normal range (Bainbridge et al. 2018). A high bacterial population in the rumen can increase the digestibility of dietary fiber and the availability of microbial protein as a protein source for dairy cows. The high energy level which is contributed by NFC sources (corn and cassava) in the treatment diet might be induced the increase of glucose fermentation by improving the levels of substrate-level phosphorylation (SLP) and electron transport phosphorylation (ETP), thereby increasing the number of rumen bacteria (Lu et al. 2019). Sulfur supplementation did not influence the rumen bacteria population and protozoa. The similar results also agree with the finding of Rosmalia et al. (2022b), who reported that sulfur supplementation in the dairy ration with the same RDP ratio did not affect the rumen bacteria population, as well as the protozoa population (Prachumchai et al. 2021; Supapong et al. 2019). The function of sulfur in rumen microbial cells is part of sulfuric amino acids (cysteine and methionine) and part of several enzymes (CoA, Co-enzyme A carboxylase) (Akib et al. 2014). Vakili et al. (2010) reported that NFC supplementation can reduce the population of methanogenic bacteria and protozoa in rumen fluid. Starch-type NFC has the potential to suppress rumen methanogenesis. In contrast, another study conducted by Firkins et al. (2007) stated that the rumen microbial population was not affected by the type of carbohydrate. The MPS found in this study ranged from 5.36 to 6.77 mg 10 mL^-1. Rumen microbes can synthesize amino acids that make up their body cells from carbohydrates, non-protein nitrogen (NPN), and organic and inorganic sulfur. The microbial synthesis process will be optimal if the carbohydrate, nitrogen, and energy are available in sufficient and balanced quantities (Hackmann and Firkins, 2015). Waldi et al. (2017) reported that the MPS in feed containing total digestible nutrient (TDN) 59.99% – 61.75% was 123.79 – 227.56 mg 20 mL^-1. The composition of the feed can cause this difference, and the feed ingredients used are also different. Rodríguez et al. (2007) stated that MPS depends on several factors, such as carbohydrate and protein sources, feed intake levels, synchronization of rumen function, recycle of rumen microbes and antinutrients in the plants consumed. Adding starch to ruminant feed can negatively affect on microbial synthesis because starch fermentation can reduce rumen pH, fiber degradation, and amino acid synthesis. Other factors supporting optimal microbial protein synthesis are nitrogen supply, VFA concentration, protein and energy degradation rates synchronization, vitamin and mineral content, and the number of rumen microbes (Pathak, 2008). The digestion process aims to break down the nutrient of feed (protein, lipids, carbohydrate) from large to simple molecules through mechanical and chemical digestion for absorption into the blood and lymph vessels (McDonald et al. 2010). Digestibility is a key factor in measuring the nutritional value of feed. The IVDMD and IVOMD in this study ranged from 68.80-69.76% and 72.27-73.15%, respectively, which indicate a moderate dairy ration in the tropical area (Lestari et al. 2015; Anzhany et al. 2022). The IVDMD and IVOMD values in this study did not differ among treatment. It might be due to the same protein content and RDP ratio in the treatment diets (Rosmalia et al. 2022c). Amount of the digestibility value of dry matter and organic matter is strongly influenced by the proportion of protein as a source of N for rumen microbes, carbohydrates as a carbon framework to support rumen microbial protein synthesis as well as an energy source for the ruminant, and crude fiber content (Hambakodu and Ina, 2019). This is also supported by a large number of similar bacterial populations in this study. Supapong et al. (2019) reported that adding sulfur as much as 2% DM to fermented total mix ration (TMR) can increase DMD followed by an increase in the bacterial population. Similar results were also reported by Promkot and Wanapat (2009), which stated that the increase in fiber digestibility cattle supplemented with sulfur at the level of 0.4% DM compared to 0.15% DM in the ration. A previous study found that the NFC ratio did not affect dry matter and organic matter digestibility (Afzalzadeh et al. 2010). In addition, Rosendo et al. (2003) stated that the digestibility of NDF in vitro in forages added with starch, inulin, and pectin was not significantly different. Meanwhile, Gao and Oba (2016) reported that the digestibility of dry matter and organic matter was higher in starch-rich diets than in disaccharide-rich carbohydrates, which was related to the rate of passage in the digestive tract.

 

CONCLUSION

In conclusion, dairy cattle ration formulation based on RDP, NFC, and sulfur synchronization produced optimum microbial activity, and protein synthesis, and in vitro digestibility in both NFC sources. Corn as an NFC produced higher protein fermentation product (NH₃) and iso-acids (iso-valerate and iso-butyrate). However, both NFC sources failed to significantly improve in microbial protein synthesis, organic matter fermentability (VFA), and digestibility. Sulfur supplementation up to 0.2% did not improve in rumen condition, fermentation product, and digestibility. It is suggested to test sulfur supplementation at a higher level further.

 

ACKNOWLEDGEMENT

The authors thank F.Y. Anggarini, L.N. Salsabila, C.M.P. Kusdiana, R. Isnaini and the Laboratory of Dairy Nutrition staff for their assistance in performing sampling and laboratory analysis. This study was supported by the Ministry of Education, Culture, Research, and Technology through the PMDSU (Accelerated Master and Doctoral Program for Excellent Bachelor) grant within contract No. 001/E5/PG.02.00PT/2022 and subcontract No. 3687/IT.L1/PT/01.03/P/B/2022.

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