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Article

Screening Dietary Fat Sources and Concentrations Included in Low- and High-Forage Diets Using an In Vitro Gas Production System

by
Saad M. Hussein
1,2,
Matias J. Aguerre
1,
Thomas C. Jenkins
1,
William C. Bridges
3 and
Gustavo J. Lascano
1,*
1
Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 29634, USA
2
Department of Medical Laboratory Techniques, Al-Kitab University, Kirkuk 36001, Iraq
3
School of Mathematics and Statistical Sciences, Clemson University, Clemson, SC 29634, USA
*
Author to whom correspondence should be addressed.
Fermentation 2024, 10(10), 506; https://doi.org/10.3390/fermentation10100506
Submission received: 12 August 2024 / Revised: 24 September 2024 / Accepted: 27 September 2024 / Published: 1 October 2024
(This article belongs to the Special Issue In Vitro Digestibility and Ruminal Fermentation Profile, 2nd Edition)
Versions Notes

Abstract

Including dietary fat can increase the energy density of diets fed to ruminants, reducing dry matter intake (DMI). Effects of different fat sources on nutrient digestion and fermentation can vary depending on dietary fat concentration and the forage-to-concentrate ratio (F:C). Therefore, this study’s objective was to screen the effects of fat sources supplemented at different concentrations to high- and low-forage diets on in vitro digestibility and fermentation. Treatments included either low forage (LF; 35%) or high forage (HF; 70%) with two fat levels (6 or 9% DM) using six different fat sources, plus control. The control diet (CON) had a basal level of fat in the diet (3% fat; 0% fat inclusion), and fat sources were added to attain 6% or 9% dietary fat and consisted of the following: Coconut oil, CO; Poultry fat, PF; Palm oil, PO; Palm kernel oil, PKO; Soybean oil, SOY; and Ca Salts, MEG. In vitro Gas Production (GP) modules were randomly assigned to treatments in a 2 × 2 × 7 factorial design and were incubated for four 24 h runs. The CO-fed module had the highest dry matter (DM) apparent digestibility (AD) (p < 0.01), followed by SOY and PF. The true DM digestibility (TDMD) and organic matter (OM) AD were the highest in CO (p < 0.01) than the other fat types. The AD for DM, OM, neutral detergent fiber (NDF), and acid detergent fiber (ADF) was higher in LF (p < 0.01). The 6% fat inclusion had a higher GP (109 vs. 103 mL ± 2.09; p < 0.03). Total volatile fatty acid (VFA) concentration was lower in different fat types than the CON and the acetate molar proportion (p > 0.01). The propionate was the lowest for the CON, which increased the acetate to propionate (A:P) ratio (p < 0.01). These results suggest that LF diets with high fat concentrations can be utilized, and different fat sources may improve DM and fiber digestibility.

1. Introduction

Adding fat to ruminant diets became common practice because of its potential to increase energy density in diets, improve palatability, and reduce feed dustiness [1]. The advantages of fat addition to dairy rations include a potential increase in energy intake for high milk production [2,3]. It can also improve rumen fermentation by optimizing the starch to fiber ratio without the risk of feeding excessive fermentable carbohydrates [4]. The use of supplemental fat has been successfully incorporated into high-forage-based diets [5,6,7]. Fat increases the density of the diet, which reduces the required intake and slows down the rate of rumen passage, allowing for more time for microbial fermentation and digestion to occur. In addition, the strategic use of fat can increase the production of volatile fatty acids (VFA) by stimulating the growth of certain microbes in the rumen that are more efficient at fermenting fiber [6,7]. Furthermore, fat can reduce the production of methane, a greenhouse gas produced during rumen fermentation, which can be beneficial for the environment. On the other hand, modifying the forage to concentrate ratio (F:C) and manipulating nutrient fractions allow for adequate nourishment in ruminants, even though high-concentrate diets showed improvement in nitrogen (N) and organic matter (OM) digestibility [8] and resulted in similar effects on rumen fermentation [9,10]. However, high grain costs, reduction in fiber digestion, and acidosis can occur because of feeding rapidly fermented non-fiber carbohydrates (NFC) in the rumen [11,12].
Cost-effective by-products from numerous industries, such as poultry industry by-products, can be utilized by ruminants. Poultry fat (PF) is a by-product of chicken processing and is extensively produced worldwide and can be a potential energy source. In contrast, soybean oil (SO) can decrease fiber digestion by inhibiting rumen microbes [13,14], whereas coconut oil (CO) might improve rumen fermentation [15,16]. Several commercial fat preparations are available as rumen bypass fats or inert fats such as Megalac (calcium salts), which is made from palm oil [17,18]. Specialty fats have been developed to minimize the detrimental effects on rumen fermentation and the risk of decreasing fiber digestion [19,20]. Thus, using these specialty fats with high saturated fats leads to minimizing adverse effects on milk fat production, rumen fermentation, and feed intake [21].
In a study conducted by Elliot et al., 1997 [22], on the effects of saturation of fat sources in steers, they reported that increasing fat sources’ saturation tended to increase the neutral detergent fiber (NDF) and acid detergent fiber (ADF) rumen digestibility. Other studies have reported no differences in ruminal or total tract digestibility of OM or fiber in lactating cows fed diets with increasing amounts of dietary fat or different sources [23,24]. In addition, [25] reported that acetate responded quadratically as the fat sources’ unsaturation degree increased. Several studies have explored various strategies for feeding fat to dairy cows [26]. However, there is limited research regarding the effects of feeding fat on the growing dairy heifers, and to what extent it can be strategically incorporated is unknown. Therefore, this study’s objective was to evaluate the effects on digestibility and fermentation, including different types of fat with different F:C ratios, using the in vitro gas production system. We hypothesized that incorporating fats in low-forage diets can improve nutrient utilization without compromising fermentation and digestibility in a gas production system. This article is based on Chapter III of [27] PhD dissertation.

2. Materials and Methods

2.1. Treatments and Experimental Design

Treatments consisted of two F:C combinations, either low-forage (LF; 35%, DM) or high-forage (HF; 70%, DM), with two dietary fat concentrations (6% or 9%) and six different fat source treatments plus control (CON). The CON had a basal level of fat in the diet containing 3% fat (0% fat inclusion), and fat sources were added to attain 6% or 9% fat and consisted of Coconut oil (CO; Nature’s oil, Streetsboro, OH, USA), Poultry fat (PF; Valley proteins, Inc., Ward, SC, USA), Palm oil (PO; Nature’s oil, Streetsboro, OH, USA), Palm kernel oil (PKO; Nature’s oil, Streetsboro, OH, USA), Ca Salts (MEG; Megalac regular), and Soybean oil (SOY; Nature’s oil, Streetsboro, OH, USA). The experiment was conducted using an in vitro ANKOMRF gas production (GP; Ankom Technology, Macedon, NY, USA) system. Treatments were randomly assigned to one of twenty-eight modules and allocated to a different module during each run to remove any module-specific differences. To allow for the CON to be compared to the other fat treatments using a factorial modeling approach, it was assumed in the statistical analysis that the CON had the same fat levels as the other treatments, 6% and 9% (not just the 3%). That resulted in a 2 × 2 × 7 factorial treatment design. The experiment design consisted of modules nested in forages, with fat and source in a randomized complete block design (run was the blocking factor), with four replicates per treatment as incubated for four 24 h runs. Each run was started with a clean module and inoculated with fresh ruminal contents collected from two multiparous cannulated Holstein cows with a body weight (BW) of 556 ± 35 kg and days in milk (DIM) numbering 120 ± 36.6.
All diets were fed to the modules as total mixed rations (TMR), and the predicted nutrient composition was determined using [28]. Dietary ingredients and chemical composition are presented in Table 1. Rations were ground using a Wiley Mill (Arthur H. Thomas Co., Philadelphia, PA, USA) through a 1 mm sieve, and 1 g of the premixed rations were placed in ANKOM F57 filter bags, sealed, and placed into the module glass bottle at the beginning of the 24 h incubation.

2.2. Module Culture Conditions

All procedures involving the surgical and animal care protocols were approved by the Clemson University Institutional Animal Care and Use Committee. Around 1800 h, the rumen contents were collected from two rumen cannulated Holstein cows fed a 50% forage:50% concentrate diet and strained through two layers of cheesecloth into a prewarmed sealed container. The filtered rumen fluid was combined from both cows and mixed with a buffer in a 1:4 ratio, homogenized together under magnetic stirrer, and purged with CO2 until inoculation into the GP modules. Also, during the time from when the rumen contents were collected to dilution and addition to modules (did not exceed 60 min), the module glass bottles were maintained at 39 °C in a water bath to minimize the cold shock of microorganisms. Pre-prepared F57 filter bags (pre-rinsed F57 filter bags in acetone for 5 min and air-dried to remove surfactant that inhibits microbial digestion) containing 1 g of the premixed rations were placed into the module glass bottles. Exactly 100 mL of diluted inoculum (20 mL inoculum + 80 mL buffer [29]) was added to each gas production module glass bottle and placed in a 39 °C shaker water bath (70 rpm; Julabo SW22, Seelbach, Germany). They were purged continually with CO2 directly into the bottle’s top until the CO2 filled the module glass bottle, and were then reattached the module to the glass bottle. The module cultures were connected to the computer by a radio frequency modem that allows for each module to communicate remotely with the computer. After calibrating the gas production sensors, we started recording data using the GP software (version 11.4) and maintained this for a 24 h of incubation, and then stopped recording data, and data were saved in an Excel spreadsheet. A 24 h time period was chosen to reflect rumen turnover rates of growing dairy heifers.

2.3. Sample Collection and Analysis

After 24 h of incubation of each run, the F57 filter bags were removed from the module glass bottles and rinsed out twice with distilled water and gently pressed to remove excess gas and water, then placed in a forced-air-drying oven for 48 h at 65 °C to determine the apparent dry matter (DM) digestibility. Following that, F57 filter bags were placed in the Ankom Fiber Analyzer and followed the procedure for determining neutral detergent fiber (NDF) to determine the true DM digestibility (when determining true digestibility, it is necessary to remove any remaining soluble fractions using a natural detergent solution; after rinsing the bags in cold tap water until the water is clear, place them in the Ankom Fiber Analyzer; Ankom technology method 3). Also, to determine the NDF apparent digestibility, feed samples were ground using a Wiley Mill (Arthur H. Thomas Co., Philadelphia, PA, USA) through a 1 mm sieve and were analyzed for DM, OM, ash, and EE [30]. For NDF and ADF [31], an ANKOM200 Fiber Analyzer (ANKOM Technology Corporation, Fairport, NY, USA) was used with heat-resistant α-amylase and sodium sulfite utilized in the NDF procedure and adjusted for ash content. Additionally, cultural contents were mixed thoroughly in the module glass bottles during sampling to ensure adequate sampling from the cultures.
Culture pH was measured and recorded after 24 h of incubation, and a 5 mL sample of culture contents was taken at the same time points for volatile fatty acids (VFA) and ammonia analysis. Culture samples (5 mL) were pipetted to 15 mL centrifuge tubes containing 1 mL of metaphosphoric acid (25%; w/v), and then these tubes were stored at −20 °C until VFA and ammonia analysis, as described by [32]. Samples were later thawed and centrifuged at 40,000× g for 30 min at 4 °C. After centrifugation, 1 mL of the supernatant was placed in a 2 mL Eppendorf microcentrifuge tube and used to analyze NH3-N, according to [33] with modifications, including reduced sample and reagent volume to accommodate the use of a 96-well plate reader. Another 0.5 mL of the supernatant was combined with 0.5 mL distilled water and 100 μL of internal standard (86 μmol of 2-ethylbutyric acid/mL) in a gas chromatography (GC) vial. GC then analyzed samples for VFA–flame-ionization detection according to the methods of [34] and injected into a Hewlett-Packard 6890 gas chromatograph (San Jose, CA, USA) equipped with a custom packed column (2 m × 0.32 cm × 2.1 mm ss; 10% SP-1200/1% H3PO4 on 80/100 Chromosorb WAW).

2.4. Statistical Analysis

All statistical analyses were conducted in SAS version 9.4 for Windows (SAS Insti-tute Inc., Cary, NC) using the MIXED procedure. Data were analyzed as a 2 × 2 × 7 factorial treatment structure. The experiment design had modules nested in forages at one level, and fat and source in a randomized complete block design at a second level (basically a split-plot type design). Forage, fat, source, forage × fat, fat × source, and forage × fat × source were considered to be fixed effects, and module (forage) and run were considered to be random effects. This resulted in the following model:
Yijk = μ + Fi + Ml(Fi) + Pj + Ck + FPij + PCjk + FPCijk + Rm + eijklm,
where Yijk = the dependent variable, μ = the overall mean, Fi = the fixed effect of forage, Ml(Fi) = the random effect of a module within forage, Pj = the fixed effect of fat, Ck = the fixed effect of source, FPij = the interaction between forage and fat, PCjk = the interaction between fat and source, FPCijk = the interaction between forage, fat, and source, Rm = the random effect of a run, and eijklm = the residual error. The PDIFF option-adjusted by the Tukey method was included in the LSMEANS statement to account for multiple comparisons. Residuals for all models were found to be normally distributed (Shapiro–Wilk test for normality). Least-squares means are presented in the tables, and evidence for statistical significance was declared at p ≤ 0.05, while trends for main effects and interactions are discussed at 0.10 ≥ p > 0.05.

3. Results and Discussion

3.1. Diet Composition and Nutrient Inputs

Diet ingredients and chemical composition values are presented in Table 1. Diets were planned and formulated to differ mainly in providing dietary fat by adding different fat sources. The dietary neutral detergent fiber (NDF) and acid detergent fiber (ADF) were lower for low-fiber (LF) diets compared to the high-fiber (HF) diets, whereas non-fiber carbohydrates (NFC) were higher for LF diets than for HF diets, as was their input because of the lower level of forage and a higher level of concentration in these diets (Table 1). The dietary ether extract (EE) concentrations increased gradually in the diets, up to 9% with different fat inclusion. The fat inclusion replaced the ground corn in the control diet of both the LF and HF diets, and that resulted in a decrease in NFC in the other different types of fat treatments. All other components of the rations were formulated to be similar between treatments.

3.2. Digestibility of Nutrients

3.2.1. Forage Effect

Apparent digestibility (AD), true dry matter digestibility (TDMD), and cumulative gas production (GP) are outlined in Table 2. The AD of dry matter (DM), organic matter (OM), NDF, and ADF were greater for the LF-fed module (p < 0.01) than for the HF-fed module. These observations are consistent with results reported in a study conducted on Holstein dairy heifers fed LF or HF diets composed of a combination of 40 or 80% CS and corn stover [35], where DM and OM AD were higher for LF compared to HF diets. Two levels of F:C diets were fed to dairy heifers by [36] and observed higher DM and OM AD for LF compared to HF diets. Similarly, higher DM and OM digestibilities for LF compared to HF diets were observed by [37]. The greater digestibility of the LF-fed module can be attributed to the greater digestibility of these diets’ ingredients [28]. Other studies have shown an increase in DM and OM AD when LF and HF diets have been fed restrictively [5,38,39]. These results did not agree with a study conducted on Holsten heifers fed low-forage (45% forage) and high-forage (60% forage) diets where DM and OM AD did not differ between LF and HF diets [40]. The AD of NDF and ADF disagreed with [41]’s findings where the ruminal digestion of NDF was improved in HF diets containing dried distiller’s grains (DDGS) in dairy cows compared with LF diets containing DDGS. They attributed that to the ability of fat from DDGS to bind in the feed particle and slowly become introduced into the rumen. Furthermore, this could be attributed to the lower pH level for the LF diet because cellulolytic bacteria are very sensitive to pH and their activity and growth start to decline under pH 6.0 [42], but the pH in the current study was similar because of the type of buffer used in the experiment. Other studies [8,37,40] observed that the ADF AD did not differ between LF and HF diets, but was in agreement with several studies where NDF AD was greater for LF diets [8,35,36].
The LF-fed module showed a higher cumulative gas production (p = 0.01) compared to the HF-fed module. The current finding agrees with [43], where they conducted an in vitro study to measure the total gas production of rumen fluid collected from non-lactating cows fed three levels of concentrate diet. They reported that a high proportion of concentrate produced the highest total gas after 24 h of incubation. They attributed that to the fact that the concentrate digestibility is faster than forage digestibility, which explains the higher total gas production observed in a high proportion of concentrate. Also, [44] reported a higher accumulated gas production as a concentrate level increased in the diet when feeding four different F:C ratios and using a gas fermentation production technique for 96 h of incubation.

3.2.2. Fat Effect

The level of fat inclusion did not show any effect on nutrients’ apparent digestibility (AD, p > 0.05, Table 2). These findings agreed with a study conducted by [45], where the DM and OM did not differ between the treatments. Rumen microbes are able to saturate fatty acids (FA) when fat levels are low in the diets, but this capacity can be exceeded at higher levels of fat, and FA can accumulate in the rumen and interfere with rumen fermentation and digestibility [28], whereas they have observed a higher AD of NDF and ADF when heifers were limit-fed a high-fat DDGS compared to a low-fat DDGS. It was suggested that the high-fat DDGS diet contains a lower starch content compared to the low-fat DDGS, which resulted in higher efficiency of utilization of fiber and improve total-tract digestion. Also, [37] observed a quadratic DM, OM, NDF, and ADF AD response to increasing levels of DDGS up to 14% inclusion in the diets. These results agreed with a study conducted by [46] using two levels of fat with no added fat or 3.3% added soybean oil in a continuous culture fermenter. They did not observe any effects on DM and ADF AD between the two diets’ two levels of fat. While [47] reported a depression in DM, OM, NDF, and ADF AD when continuous culture fermenters were fed high soybean oil compared to low soybean oil, feeding excess FA has been reported to depress fiber digestibility [18,48]. Also, the dietary polyunsaturated fatty acid as in soybean oil has been related to limiting the growth of fiber-digesting bacteria, which reduce ruminal fiber digestibility [49,50,51]. However, [52] reported no effects on AD when heifers were limit-fed DDGS with ad libitum grass hay. They related that to feeding grass hay as ad libitum, which resulted in a slightly different limit feed program than the typical one.
The cumulative gas production was decreased as the level of fat inclusion increased in the diet (p = 0.03). The supplementation of plant oil decreased gas fermentation production in a study conducted by [44] using two different plant oil inclusion levels. Also, [53] reported that when ruminants receive diets with a fat content higher than 7% of DM, fiber digestion could be restricted, which might explain the current finding.

3.2.3. Source Effect

The apparent digestibility (AD) of DM, OM, NDF, ADF, and TDMD were affected by the type of fat with greater DM AD for the CO-fed (p < 0.01), followed by SOY, PF, CON, PKO, and PO-fed (Table 3). Also, TDMD and OM AD were the highest with the CO-fed module (p < 0.01), followed by all MEG, SOY, PF, PO, CON, and PKO-fed modules. Furthermore, the NDF AD was higher in MEG and CO-fed modules followed by PO, SOY, PF, CON, and PKO-fed modules, whereas the ADF AD was similar between fat types except for the PKO-fed module with the lowest value. These observations are consistent with results reported in a study conducted by [22] on the effects of steers’ saturation of fat sources. It has been reported that increasing saturation of fat sources (tallow, partially hydrogenated tallow, hydrogenated tallow, blend of hydrogenated tallow and hydrogenated fatty acids, and hydrogenated fatty acids) tended to increase the NDF and ADF digestibility in the rumen. Several other studies have reported no differences in ruminal or total tract digestibility of OM or fiber in lactating cows fed diets with different fat sources [23,24]. It has been reported in an in vitro study that the CO, which contains highly saturated medium-chain fatty acids, had no adverse effect on DM digestibility in the in vitro gas fermentation production technique [44]. That is in agreement with other studies on CO’s effect on swamp buffalo by the same group [16].
A study conducted by [46] using 3.3% added SO in a continuous culture fermenter did not observe any effects on DM and ADF AD compared to the control diet, whereas [47] reported depression in DM, OM, NDF, and ADF AD when continuous culture fermenters were fed high SO compared to low SO. A study by [47] stated that dietary polyunsaturated fatty acids had been shown to depress fiber AD by limiting the growth of fiber digestion bacteria [49]. This finding is common in the literature [18]. In addition, [54] conducted a study on steers fed treatments consisting of no added fat, 3.5% tallow, and soybean oil soap stock. They reported that adding fat did depress DM and fiber digestibility. Also, [55] investigated the effects of feeding calcium salts of poultry oil on dairy cows compared to a palmitic acid-enriched fat and a mix between the two. They observed that the fiber and protein digestibility were similar between treatments. They concluded that even though the calcium salts of poultry oil improved dairy cows’ production but decreased feed efficiency. A literature review reported by [56] on tallow digestibility compared to other fat sources found that only tallow and calcium salts of palm FA had numerically higher digestibility than the other fat sources examined. Furthermore, [57] reported an increase in NDF digestibility when Ca soap’s level increased in the diet. It has been suggested that the higher apparent total tract digestibility of NDF in cows-fed Ca-LCFA was related to an increase in post-ruminal degradation [58]. The digestibility of ADF under bypass fat addition may be either increased [59] or not affected [60,61]. It has been reported that the ADF digestibility varies depending on the level of fat addition, with no effect at a low fat level [62]. A study by [63] reported that bypass fat did not influence buffaloes’ cellulose digestibility. Also, [64] reported that hemicellulose digestibility was improved with the addition of Ca-LCFA and caused an increase in NDF and a decrease in ADF digestibility in dairy cows.
Cumulative gas production was affected by the different types of fat, with the highest value for the CO-fed module and SOY-fed module (p = 0.03), followed by all of the PF-fed module, CON-fed module, and PO-fed module, and then the MEG-fed module and PKO-fed module. These results did not agree with [16,44], as they reported a reduction in gas production when CO was included in the diets. They attributed that to the negative effect of medium-chain fatty acids on the fermentation, as they are small enough to be readily dissolved and disrupt the cell membranes and inhibit enzymes involved in energy production and lead to the microbial death of cells [65].

3.3. Characteristics of Fermentation

3.3.1. Forage Effects

Culture VFA profile, NH3-N, and pH are shown in Table 4. The total VFA concentration was lower for the LF-fed module than for the HF-fed module (p < 0.01), mainly because of the lower acetate molar proportion for the LF-fed module compared to the HF-fed module. In contrast, propionate and butyrate molar proportions were higher for the LF-fed module than the HF-fed module (p < 0.01). As a result, the acetate:propionate ratio was lower in the LF-fed module than in the HF-fed module (p < 0.01). The lower total VFA concentration for LF-fed fermenters did not agree with in vitro and in vivo studies [36,66]. In addition, [67] concluded that the main factor influencing VFA concentration is the interaction between pH and F:C in the diets. Also, [32] stated that the VFA concentrations were higher in LF than HF when pH was affected by F:C.
In the current study, the pH was similar between the LF-fed module and the HF-fed module and is mainly related to the type of buffer used in this study to keep the culture in the same pH level for 24 h of incubation. While the greater acetate molar proportion for HF-fed fermenter is consistent with previous studies [36,37,68,69], acetate results of structural carbohydrate fermentation by cellulolytic bacteria and these bacteria can be inhibited by lower NDF inputs, as in the present study, which may explain the lower acetate molar proportion for LF-fed fermenters [70]. Several studies showed that the F:C ratio did not affect propionate and butyrate [37,69,71]. Cultural pH was similar between the LF-fed module compared to the HF-fed module. The pH values did not agree with a study conducted using Rusitec fermenters, as they reported a higher pH in HF-fed fermenters than for LF-fed fermenters [68]. The NH3-N concentration was lower for the LF-fed module than for the HF-fed module (p < 0.01). These findings agree with several studies using different F:C ratios in continuous culture fermenters [66,67,68]. The lower NH3-N in the LF-fed module than in the HF-fed module could be due to ammonia for AA’s de novo synthesis.

3.3.2. Fat Effects

The fat inclusion level in the diets decreased the total VFA concentrations with a lower value for the 9% fat-fed module than the 6% fat-fed module (p < 0.01, Table 4). In contrast, the acetate, propionate, butyrate molar proportions, and the acetate:propionate ratio were not affected by fat inclusion in the diets. Rumen fermentation is not affected when fat levels are low in the diets because rumen microbes are able to saturate FA, but this capacity can be exceeded at higher levels, and FA can accumulate in the rumen and interfere with rumen fermentation [28]. The different levels of fat inclusion decreased the culture pH (p = 0.01). While [72] reported a similar rumen pH between treatments as DDGS increased in the diets, in contrast, [73] observed a linear decrease in rumen pH as DDGS increased in the diets, and they attributed that to the F:C ratio. Ammonia concentration was increased as the fat inclusion increased in the diets (p = 0.04). Other studies [37,73] observed similar results, and they attributed that to the lower ME intake with the addition of DDGS. Therefore, the microbial capacity to assimilate amino acids and ammonia was negatively affected and NH3 accumulated in the rumen [28]. Also, [74] attributed the higher NH3-N concentration as fat included in the diets is due to the greater proteolytic bacteria.

3.3.3. Source Effects

The different types of fat in the diets decreased the total volatile fatty acid (VFA) concentrations in the modules compared to the CON-fed module (p < 0.01, Table 5). A study conducted by [22] reported that the total VFA concentration decreased when different fat sources were fed compared to the control diet. Also, [44] reported a lower total VFA concentration in the in vitro gas production technique after 48 h incubation with a CO diet. They attributed that to the negative effect of medium-chain fatty acids on the fermentation. Also, the higher total VFA concentration for CON-fed fermenters could be related to the pH, as it is the main factor influencing VFA concentrations [67]. In the current study, the pH was lower for the CON-fed module compared to the CO-fed module and PF-fed module. A study by [25] reported that the acetate responded quadratically as the fat sources’ unsaturation degree increased (tallow, partially hydrogenated tallow, and animal–vegetable fat). In addition, [22] reported a decrease in acetate’s molar proportion when different saturated fats (tallow, partially hydrogenated tallow, hydrogenated tallow, blend of hydrogenated tallow, and hydrogenated fatty acids, and hydrogenated fatty acids) were fed and increased linearly as saturation increased.
In contrast, the propionate molar proportion was higher for the CO-fed module and PF-fed module (p < 0.01) compared to the CON-fed module and other fat type treatments. The propionate finding did not agree with the study conducted by [44], as they reported a lower propionate in the CO diet after 48 h incubation. Also, in the study by [25], Holstein heifers were fed a different degree of fat saturation (tallow, partially hydrogenated tallow, and animal–vegetable fat). Also, [22] reported a linear decrease in propionate as saturation increased. As a result of the increase in propionate molar proportion, the acetate:propionate ratio was the lowest in the CO-fed module and PF-fed module (p < 0.01) compared to the CON-fed module and the other fat types treatments. Some studies have reported that feeding fat can decrease the acetate:propionate ratio [22,25] or leave it unchanged [75]. They related the decrease in the acetate:propionate ratio to the reduction in ruminal NDF AD, which is not the case in the current study. Ruminal fermentation has been frequently shifted to greater propionate in cows fed fats such as tallow, yellow grease, or animal-vegetable blends, and has resulted in lower acetate:propionate ratios [21,62,76,77]. Butyrate molar proportion was lower in the CON-fed module compared to the different types of fat treatments. A study by [52] suggested that the differences in starch contents and intake are the reason behind the shift in VFA concentrations and the decrease in acetate and increase in propionate. They also suggested that higher propionate is related to more energy-efficient and rumen fermentation in heifers fed DDGS diets [73]. There is less methane and carbon dioxide production in propionate as compared with acetate [78].
Cultural pH was lower for the CON-fed module compared to the different types of fat-fed module. A study by [79] stated that the drop in pH in high-starch diets is common in the literature. The inclusion of different fat sources in the diets increased the cultural pH, with the highest pH values being observed in the CO-fed module, followed by the PF-fed module compared to the CON-fed module. That agrees with a study conducted by [22], where they reported an increase in ruminal pH as different saturated fat were fed, and they attributed that to the lower fermentable carbohydrate content in these diets. The NH3-N concentration was similar between the different types of fat compared to the CON-fed module, except for the MEG-fed module, which had the highest NH3-N concentration. A study by [22] reported a linear increase in NH3-N concentrations as the degree of saturation increased (tallow, partially hydrogenated tallow, hydrogenated tallow, blend of hydrogenated tallow and hydrogenated fatty acids, and hydrogenated fatty acids), and they suggested that the dietary triglycerides became more unsaturated, and the ruminal protein digestion inhibited. These results could be related to better synchrony between N and energy availability for a microorganism’s activity. These results agreed with previous studies where the ruminal NH3-N concentrations were not affected by supplemental fat or fat sources [25,80,81].

4. Conclusions

Screening different fat sources with varying concentrations of inclusion in low- and high-forage diets using a gas production system showed differential effects on culture fermentation. These results show that the LF-fed modules consistently resulted in higher nutrient utilization and the apparent digestibility of most nutrients, while the level of fat inclusion had no detrimental impact on nutrient digestibility. Results from this study demonstrate that the inclusion of CO resulted in the highest digestibility of nutrients and can significantly improve dry matter digestibility when used in high-fat diets. More saturated types of fat, such as PF and CO, shifted the fermentation profile towards a more propionate abundant environment with the respective change in A:P. Therefore, we can conclude that a low-forage diet (35%) with fat inclusion of up to 6% from different saturated fat sources can be successfully included in isocaloric rations for ruminants without a negative effect on nutrient digestibility and fermentation characteristics.

Author Contributions

Conceptualization, S.M.H. and G.J.L.; methodology, S.M.H., M.J.A., T.C.J., W.C.B. and G.J.L.; formal analysis, S.M.H., W.C.B. and G.J.L.; investigation, S.M.H., M.J.A., T.C.J. and G.J.L.; writing—original draft preparation, S.M.H. and G.J.L.; writing—review and editing, S.M.H., M.J.A., T.C.J. and G.J.L.; visualization, S.M.H. and G.J.L.; supervision, G.J.L.; project administration, G.J.L.; funding acquisition, G.J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This project was supported by NIFA/USDA, under project number SC-1700551.

Institutional Review Board Statement

The animal study protocol was approved by the Clemson University Committee on Animal Use (AUP2019-074).

Informed Consent Statement

Not applicable.

Data Availability Statement

Dataset available on request from the authors.

Acknowledgments

Approved as Technical Contribution No. 7363 of the Clemson University Experiment Station. The authors thank Valley Proteins, Inc., Ward Division, SC, USA, for the donation of the dietary poultry fat. Appreciation is also extended to members of the Clemson Ruminant Nutrition Research Team for laboratory assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Ingredient and chemical composition of low (LF)- and high (HF)-forage diets containing unsaturated fat sources with different fat concentrations (3%, 6%, 9% DM) fed to the in vitro gas production system.
Table 1. Ingredient and chemical composition of low (LF)- and high (HF)-forage diets containing unsaturated fat sources with different fat concentrations (3%, 6%, 9% DM) fed to the in vitro gas production system.
Fat % in the Diet
Ingredient, 1 %Forage3%6%9%
      Coastal hayLF5.005.005.00
HF20.020.020.0
      Corn silageLF30.030.030.0
HF50.050.050.0
      Ground cornLF51.846.440.8
HF24.418.612.6
      Soybean meal (SBM)LF11.213.716.4
HF3.606.339.20
      Mineral mixLF2.002.002.00
HF2.002.002.00
      Fat inclusionLF0.002.805.79
HF0.003.046.19
Chemical composition
      DM %LF91.190.992.0
HF91.991.792.0
      OM, %LF95.395.595.1
HF93.994.194.0
      CP, %LF12.013.814.1
HF9.411.212.0
      NDF, %LF22.720.220.5
HF37.234.335.2
      ADF, %LF11.411.412.0
HF20.419.821.9
      EE, %LF3.325.588.59
HF3.085.488.21
      NFC, 2 %LF57.255.952.0
HF44.243.138.6
      Ash, %LF4.634.504.86
HF6.045.905.95
      TDNLF77.180.783.8
HF69.472.774.8
      ME, 3 Mcal/KgLF2.812.943.06
HF2.532.652.73
1 All diets were ground to 1 mm. 2 Non-fiber carbohydrates (NFC) = 100 − (Crude Protein (CP) + Ether Extract (EE) + Neutral Detergent Fiber (NDF) + Ash). 3 Metabolizable Energy (ME) calculated according to [28] using Total Digestible Nutrients (TDN) values as reported by Cumberland Valley Analytical Services, Inc., Waynesboro, PA, USA. ME = TDN × 0.04409 × 0.82. To represent better the increase in energy as fat increased in the diets, another modified equation from [28] was used. ME = (TDN × 4.409 × 1.01 − 0.45) + (0.0046 × (EE − 3) × 0.82.
Table 2. Nutrient apparent digestibility of in vitro gas production system fed low (LF)- and high (HF)-forage diets containing different fat concentrations (6% and 9% DM).
Table 2. Nutrient apparent digestibility of in vitro gas production system fed low (LF)- and high (HF)-forage diets containing different fat concentrations (6% and 9% DM).
ForageFat % p-Value
Digestibility, %LFHF6%9%SEF:CFat
DM54.647.351.250.80.39<0.010.10
TDMD80.366.773.473.60.49<0.010.31
OM78.964.671.771.90.21<0.010.21
NDF66.249.958.158.20.54<0.010.78
ADF62.642.553.152.20.73<0.010.17
GP 1 mL1111011091032.090.010.03
1 Cumulative gas production in mL; gas pressure was converted to mole using the ideal gas law, n = p (V/RT), and then converted to milliliter using Avogadro’s law, gas produced in mL = n × 22.4 × 1000.
Table 3. Nutrient apparent digestibility of in vitro gas production system fed diets containing different fat sources.
Table 3. Nutrient apparent digestibility of in vitro gas production system fed diets containing different fat sources.
Fat Type * p-Value
Digestibility, %CONCOPFPOPKOMGSOYSEType
DM50.6 c54.5 a50.6 c49.5 d50.1 cd49.7 d51.8 b0.48<0.01
TDMD72.7 d76.8 a73.0 cd72.9 cd71.2 e74.5 b73.6 c0.56<0.01
OM71.0 d75.4 a71.2 cd71.2 cd69.4 e72.7 b71.8 c0.35<0.01
NDF58.1 b59.2 ab58.0 b59.7 ab52.2 c60.8 a58.9 ab0.86<0.01
ADF53.1 a53.6 a53.1 a54.4 a45.7 b55.0 a53.5 a1.08<0.01
GP 1 mL110 ab114 a109 ab101 ab99.1 b100 b113 a5.070.03
1 Cumulative gas production in mL; gas pressure was converted to mole using the ideal gas law, n = p (V/RT), and then converted to milliliter using Avogadro’s law, gas produced in mL = n × 22.4 × 1000. * Means in the same row, followed by different superscripts (a, b, c, d, and e), are significantly different (p < 0.05).
Table 4. Volatile fatty acids, NH3-N, pH, and GP of in vitro gas production system fed low (LF)- and high (HF)-forage diets containing different fat concentrations (6% and 9% DM).
Table 4. Volatile fatty acids, NH3-N, pH, and GP of in vitro gas production system fed low (LF)- and high (HF)-forage diets containing different fat concentrations (6% and 9% DM).
ForageFat % p-Value
Culture FermentationLFHF6%9%SEF:CFat
Total VFA, mM73.177.780.969.90.66<0.01<0.01
VFA, mol/100 mol
Acetate56.366.160.961.40.32<0.010.19
Propionate27.921.924.924.90.24<0.010.94
Butyrate15.911.914.213.70.27<0.010.17
Acetate:propionate2.093.062.592.560.03<0.010.37
pH 6.606.616.626.590.010.840.01
NH3-N, mg/dL8.7012.310.110.90.33<0.010.04
Table 5. Volatile fatty acids, NH3-N, pH, and GP of in vitro gas production system fed diets containing different fat sources.
Table 5. Volatile fatty acids, NH3-N, pH, and GP of in vitro gas production system fed diets containing different fat sources.
Fat Type * p-Value
Culture FermentationCONCOPFPOPKOMGSOYSEType
Total VFA, Mm89.6 a82.8 b68.9 c69.4 c69.1 c70.3 c71.2 c1.20<0.01
VFA, mol/100 mol
Acetate67.7 a56.5 e58.2 d60.3 c61.5 bc62.2 b62.0 b0.58<0.01
Propionate20.8 d29.0 a26.8 b24.4 c24.8 c23.9 c24.5 c0.45<0.01
Butyrate11.5 c14.5 ab15.0 a15.2 a13.7 b14.0 ab13.5 b0.52<0.01
Acetate:propionate3.29 a2.00 d2.25 c2.56 b2.63 b2.69 b2.60 b0.06<0.01
pH 6.56 b6.62 a6.63 a6.61 ab6.62 a6.59 ab6.61 ab0.020.29
NH3-N, mg/Dl10.6 b10.8 b10.1 b10.2 b10.3 b11.6 a10.2 b0.440.01
* Means in the same row, followed by different superscripts (a, b, c, d, and e), are significantly different (p < 0.05).
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Hussein, S.M.; Aguerre, M.J.; Jenkins, T.C.; Bridges, W.C.; Lascano, G.J. Screening Dietary Fat Sources and Concentrations Included in Low- and High-Forage Diets Using an In Vitro Gas Production System. Fermentation 2024, 10, 506. https://doi.org/10.3390/fermentation10100506

AMA Style

Hussein SM, Aguerre MJ, Jenkins TC, Bridges WC, Lascano GJ. Screening Dietary Fat Sources and Concentrations Included in Low- and High-Forage Diets Using an In Vitro Gas Production System. Fermentation. 2024; 10(10):506. https://doi.org/10.3390/fermentation10100506

Chicago/Turabian Style

Hussein, Saad M., Matias J. Aguerre, Thomas C. Jenkins, William C. Bridges, and Gustavo J. Lascano. 2024. "Screening Dietary Fat Sources and Concentrations Included in Low- and High-Forage Diets Using an In Vitro Gas Production System" Fermentation 10, no. 10: 506. https://doi.org/10.3390/fermentation10100506

APA Style

Hussein, S. M., Aguerre, M. J., Jenkins, T. C., Bridges, W. C., & Lascano, G. J. (2024). Screening Dietary Fat Sources and Concentrations Included in Low- and High-Forage Diets Using an In Vitro Gas Production System. Fermentation, 10(10), 506. https://doi.org/10.3390/fermentation10100506

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