Blood Metabolites and Feed Utilization Efficiency in Thai-Native-Anglo-Nubian Goats Fed a Concentrate Diet Including Yeast Fermented Palm Kernel Cake Instead of Soybean Meal

Feed is the most expensive component in goat production. Hence, lowering it is crucial to increasing producer profitability. The microbial community in rumen is vital for nutritional digestion and absorption in ruminants. Live yeast and yeast-based products generated from the strain Saccharomyces cerevisiae (commercial strain) are actively being used and investigated. The purpose of this study was to investigate the effects of substituting soybean meal (SBM) in concentrate diets with yeast-fermented palm kernel cake protein (YFPKCP) on dry matter intake, digestibility, blood markers, and nitrogen balance. Five crossbred Thai Native-Anglo-Nubian goats (50% Thai Native goats with 50% Anglo-Nubian goats) weighing an average of 27 ± 2 kg were randomly allocated to one of five diets using a 5 × 5 Latin square design: 0, 25, 50, 75, and 100% YFPKCP replacement for SBM. Plicatulum hay (Paspalum plicatulum Michx.) was provided ad libitum. There were no significant differences in dry matter (DM) intake among treatments, but the apparent digestibility of DM, crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) were affected (p < 0.05) by including YFPKCP in diets. They also tended to be slightly lower for goats fed the diet containing 100% YFPKCP replacement for SBM compared to other treatments. Ruminal pH, ammonia-nitrogen (NH3-N), blood glucose, and packed cell volume were equivalent among treatments. On the other hand, replacement YFPKCP reduced digestibility and N absorption by up to 75% (p < 0.05). Furthermore, there was no difference in total volatile fatty-acid concentration among goats fed YFPKCP as a substitute for SBM. According to the results of this study, the level of YFPKCP in the concentrate replacement of SBM for goats fed plicatulum hay should be 75%.


Introduction
The meat-goat market has lately grown in popularity [1], presenting a host of new opportunities for diversifying farm earnings. However, because feed is the most expensive component of goat production, lowering it is crucial to enhancing producer earnings. Feeds that are both cost-effective and easy to handle will soon be required for meat goat producers. Oil palm (Elaeis guineensis Jacq.) is widely accessible and belongs to a well-developed oilproducing industry [2]. Palm kernel cake (PKC) is a by-product of palm oil production that is abundant in Southeast Asia, Indonesia, Malaysia, and the southern part of Thailand. Palm kernel cake, also known as palm kernel meal, has demonstrated to be an excellent feed element for a variety of ruminants and is widely accessible in tropical countries [3,4]. PKC has ametabolizable energy (ME) content of 20.6 MJ/kg [5]. The nutritional value of forages for ruminant feeding has been tested utilizing the procedure of protein enrichment of animal feed employing microorganisms in a semi-solid culture. Punj [6] discovered that, depending on the method used to extract the oil, palm kernel meal contains 12-23% DM of crude protein and has an in vitro dry matter digestibility of 70-80% [7]. However, because of its high neutral detergent fiber (NDF) content (60-70% NDF) and low palatability, PKC's application is limited [5,6]. In ruminant nutrition, introducing microbial fermentations to the feed, such as a Saccharomyces cerevisiae culture, has become a regular practice and has caused beneficial changes in the activity and numbers of rumen microbes. S. cerevisiae fermentation products were proven to be especially beneficial during times of stress and disease, lowering rumen pH oscillations and enhancing dry matter intake [8]. This might be related to alterations in the rumen microbial community which would result in changes in ruminal VFA production [9].
Furthermore, the effect of S. cerevisiae fermentation products on dairy cows can enhance milk production and weight gain in growing cattle [10]. When yeast was added to dairy lactating cows, it enhanced milk quality [11], altered feed intake [12], and improved immunological function [13]. Cellulolytic and lactate-utilizing bacteria in the rumen stimulation increased fiber digestion, and greater microbial protein flow from the rumen are all frequent production responses attributed to yeast that may be advantageous for feedlot cattle fed high-grain diets [14]. The inclusion of yeast culture in animal nutrition has a positive effect that improves non-starch polysaccharide degradation which can increase the energy concentration and the release of nutrients [15]. Ruminant feeding of S. cerevisiae products can modify the rumen environment, increasing populations of microorganisms associated with fiber digestion [16], lactic acid utilization, and ruminal pH [17]. This may contribute to reducing the cost of production in ruminant systems using low-quality animal feeding by-products as major components, replacing the much more expensive cereal grain and protein source. However, the application of yeast fermented palm kernel cake (YFPKCP) as a SMB substitute has not yet been evaluated. The aim of this experiment was to examine how adding YFPKCP in replacement of SBM in a concentrate diet affected feed utilization and rumen fermentation characteristics, volatile fatty acid profiles, and the nitrogen balance of Thai-native-Anglo-Nubian goats.

Dietary Preparation of Yeast Fermented Palm Kernel Cake (YFPKCP)
To simulate yeast, 20 g of S. cerevisiae (Berry Yuker Specialities Company Limited, Bangkok, Thailand) was mixed with 100 mL of water and 20 g of sugar and kept at room temperature (25 ± 2 • C) for 2 h with a pH of 4.5-5.0 before being flushed with oxygen for 18 h. S. cerevisiae was mixed with agar medium and molasses: urea: water at a ratio of 24:72:100 (a culture medium: yeast inoculation), and incubated for 60 h at room temperature ( [18,19]). The palm kernel cake was obtained in the south of Thailand and inoculated with S. cerevisiae before being ensiled in a plastic, sealed container under anaerobic conditions. The samples were sun-dried for three days (25-32 • C) before being analyzed for their component and chemical content (Table 1).

Animals, Experimental Design, and Feeding
The study was conducted under approval record No. 48/2017 of the Animal Ethics and Care issued by Prince of Songkla University. The goats used in this experiment were obtained from the Animal Farm Department, Faculty of Natural Resources, Prince of Songkla University. Because the Animal Farm Department has a limited number of animals with the same condition (breed, BW, age, etc.), it is difficult to offer 10 animals for the current study. Hence, only a few animals were employed. The study, on the other hand, was carried out with caution and control in order to avoid any unintentional mistakes produced by humans, animals, or the environment. Five male crossbreed Thai native-Anglo-Nubian goats (50% Thai Native goats with 50% Anglo-Nubian goats) with an average live weight of 27 ± 2 kg were randomly assigned to one of five meals (0, 25, 50, 75, and 100% YFPKCP substitution for soybean meal (SBM)), respectively, using a 5 × 5 Latin square design. Each goat was housed in a metabolism cage with access to water and mineral blocks at all times. During each period, all goats were fed a 2% BW (DM as fed) concentrate diet and plicatulum hay (PH, Paspalum plicatulum, Michx.) was provided ad libitum, with 10% PH refusals allowed ( Table 2). The goats were fed twice daily at 8:00 a.m. and 4:00 p.m. Each experimental period lasted for 21 days. The first 15 days were utilized to adjust to the new treatment and monitor feed intake, while the final 6 days were used to determine digestibility using the total collection method [20]. This included 5 days of total feces and urine collection, and the final day for collection of rumen fluid and blood.

Sample Collection and Sampling Procedures
During the last 6 days of each period of the digestibility trial, rumen fluid was taken from all animals using a stomach tube (0.3 cm internal diameter with a length of 1.5 m) at 0 and 4 h post-feeding to verify the effect of rumen fermentation after feeding, and for examination of pH, NH 3 -N, rumen microbe, and volatile fatty acid (VFA) content at the end of each period. The pH of rumen fluid was determined using a portable pH and temperature meter (Hanna Instruments HI 8424 microcomputer, Singapore). The NH 3 -N evaluation was made using 5 mL of sulphuric acid (H 2 SO 4 ) mixed with 45 mL of rumen fluid. The solution was centrifuged at 16,000× g for 15 min for NH 3 -N analysis by steam distillation with a Kjeltech Auto 1030 analyzer (Tecator, Hoganiis, Sweden). VFAs analysis was performed using high-performance liquid chromatography [21]. Blood samples were drawn from the jugular vein at 0 and 4 h after feeding and placed in tubes containing 12 mg of EDTA. Plasma was separated by centrifugation at 2500× g for 15 min and tested for blood urea nitrogen (BUN) [22], blood glucose, and packed cell volume (PCV) using commercial kits (No. 640, Sigma Chemical Co., St. Louis, MI, USA).
Feeds and fecal samples were collected every period to be chemically analyzed for DM, nitrogen, ash, CP, and acid detergent fiber (ADF), following the method of AOAC [23]. The detergent fiber standards technique was used to estimate the neutral detergent fiber (NDF) in samples. Urine samples were tested for urinary N using the AOAC's Kjeldahl technique [23], and N utilization was estimated. Rumen fluid was diluted in a solution of 10% formalin in sterilized 0.9% saline to dilution, and the counts of bacteria, protozoa, and fungus were assessed using a haemocytometer and the direct counting microscopic technique (Boeco, Hamburg, Germany).

Analyses Statistical
The statistical analysis accounted for the 5 × 5 Latin square design using the GLM and treatment means, procedures of SAS [24] (SAS Inst. Inc., Cary, NC, USA). The following model was used to analyze the data: where: Y ijk , observation from goat j, receiving diet i, in period k; µ, the overall mean, M i , the effect of the different treatments (i = 1, 2, 3, 4, 5), A j , the effect of animal (j = 1, 2, 3, 4, 5), P k , the effect of the period (k = 1, 2, 3, 4, 5), C l , the effect of collection time, B m the effect of interaction between treatments and collection time, and ε ijk the residual effect.
To find differences between means, the treatment means were statistically analyzed using Duncan's Multiple Range Test [25], and significance was defined at a p value of 0.05 for differences in means and trends. Linear and quadratic effects were used to determine orthogonal polynomials for diet responses. Table 3 shows the effects of varying degrees of SBM substitution with YFPKCP in goats. Roughage and concentrate consumption were comparable between treatments (p > 0.05) with the total intake ranging from 20.54 to 27.19 g/kg BW 0.75 . It was found that the level of YFPKCP in the concentrate replacement of SBM had no effect on total DMI in growing goats. The apparent digestibility of DM, OM, CP, NDF, and ADF was lowered when SBM was replaced with 100% YFPKCP. Replacement SBM by YFPKCP at 25-75% had no effect on DM, OM, or CP apparent digestibility when compared to 0 percent (SBM only). Feeding YFPKCP at 75% enhanced NDF and ADF apparent digestibility compared to the other levels. The increase of YFPKCP in the diet was because of the associated high fibrous fraction (ADF and ADL) and EE content. The YFPKCP has a high level of nonstarch polysaccharides. The introduction of 100% YFPKCP (5.82% EE) reduced digestibility, particularly for ADF digestion and rumen microbial fermentation [26,27]. Feed intake, digestibility, fermentation process, and bacterial proliferation in the rumen were all affected by the fat content of up to 5% in a diet [28]. Furthermore, the lower digestibility of using 100% YFPKCP instead of SBM may have contributed to a lower true protein in the diet. Saxena et al. [29] found a true protein supplementation to be more effective than NPN supplementation. Similarly, Huntington and Archibeque [30] found that ruminants fed true protein had higher protein digestibility than those fed urea or NPN.

Rumen Fermentation Characteristics
The rumen parameters of temperature, pH, NH 3 -N, and BUN were all measured (Table 4). Overall, ruminal temperature and pH were within the ranges considered to be favorable for microbial fiber and protein digestion [31]. YFPKCP diets had no influence on ruminal NH 3 -N levels, which varied from 14.14 to 16.71 mg/dL. Ruminal NH 3 -N concentrations exceeded 5-8 mg/dL which is the optimal amount for microbial protein production [32]. Nitrogen supplies from protein breakdown in the rumen are combined with free amino acids, peptides, and ammonia from amino acid deamination to produce microbial protein synthesis [33,34]. The use of ammonia in the rumen is inextricably linked to the availability of carbohydrates [35]. The yield of microbial protein synthesis generated in the rumen is maximized when the ratio of available energy or fermentable organic matter to protein or nitrogen is adjusted [36]. Meanwhile, at 4 h post-feeding, the ammonia concentration in the rumen tends to increase due to protein degradation by the rumen bacteria. The NH 3 -N produced in the rumen is either used by the rumen bacteria for maintenance and growth or transformed into urea in the liver and excreted in the urine when the supply exceeds the requirement.

Ruminal Volatile Fatty Acid (VFA) Profiles
The molar ratios of the VFA profile were unaffected by the YFPKCP replacement SBM (Table 5). Total VFA, acetic acid, propionic acid, and butyric acid had mean values of 75.0 to 79.2 mM, 61.7 to 62.6 mM, 26.2 to 27.1 mol/100 mol, and 10.7 to 11.5 mol/100 mol, respectively, demonstrating that fermenting palm kernel cake with yeast may improve quality and make it comparable to SBM [37]. It was postulated that yeasts might help fungi to colonize lignin structures. The breakdown of cellulose filter paper by Neocallimastix frontalis was similarly accelerated in the presence of live yeast cells in the same studies. This impact has several routes of action, one of which is the source of thiamine, a nutrient required by rumen fungi for zoosporogenesis. It has also been proven that some yeast strains can increase the proliferation and activity of fibrolytic bacteria.

Rumen Microorganisms
When the amount of YFPKCP replacement SBM was increased, bacteria and fungi levels increased significantly (p < 0.05; Table 6). Enhanced ammonia incorporation into microbial protein and increased flow and absorption of amino acids, as well as altered endogenous nitrogen metabolism, improved nitrogen intake consumption in ruminants fed yeast culture. In several experimental meals, adding yeast to the control ration enhanced NH 3 -N levels. Protozoal populations, on the other hand, were not statistically different (p > 0.05). Protozoal populations at 0 and 4 h post feeding, as well as total protozoal populations, tended to decrease (p = 0.07) in goats fed 25-100% YFPKCP replacement for SBM, compared to goats not fed YFPKCP. This is because fatty acids in oil in the pulp of oil palm kernels are poisonous. According to Galbraith and Miller [38], longchain fatty acids are more dangerous to microbial cells than short-chain fatty acids. In comparison to other groups, the protozoal population in palm cake kernel treated cattle and sheep tended to decrease [39]. Thus, the inclusion of PKC in diets can reduce the protozoal population, altering the ruminal environment by lowering protozoal numbers while indirectly enhancing bacterial numbers and activity. Moreover, Garcia et al. [40] discovered that supplementing live yeast cells in concentrated feed using corn as the major energy source can lower entodinidae and holotrichidae populations in goats. It is possible that live yeast is attached to the oil palm kernel meal in yeast fermented palm kernel meal.

Glucose Concentration and Red Blood Cell Volume
Because yeast culture enhanced microbial activity and increased nitrogen absorption into microbial protein, the addition of YFPKCP to diets had no effect on BUN concentration (p > 0.05). Preston et al. [41] discovered that BUN levels were related to rumen ammonia production and were significantly correlated with edible protein consumption. There was no change in blood glucose levels between the YFPKCPs at 0 and 4 h after feeding (p > 0.05). The blood glucose levels in the goats varied from 63.92 to 65.00 mg/dL, with normal values being 50-75 mg/dL [42] (Table 7). Many factors influence blood glucose levels in animals, including physiological state [43], disease [44], and sample time [45,46]. According to Mahardika et al. [47], blood glucose levels rose 3-4 h after a meal. This was because propionic acid, a precursor to glucose production, was highest in the rumen 3 h after feeding. PCV at 0 and 4 h after feeding did not differ significantly across groups (p > 0.05), with a range of 28.27-29.15%. Rasedee et al. [48] found that PCV values varied depending on the level of diet given to the animals. The PCV value of dairy cows fed 1.75% BW was higher than that of cows fed 1% BW. Furthermore, PCV values in dairy cows (35.91%), buffaloes (38.37%), and cattle (30.37%) were substantially different (p < 0.05) depending on the animal species.

Nitrogen Utilization
Total nitrogen intake was not different in goats fed various YFPKCPs (p > 0.05) ( Table 8). The concentration diet with no YFPKCP had higher urine nitrogen excretion than the other YFPKCP inclusions (p < 0.01). This could be due to the amount of protein that has increased CP digestion [49]. Tamminga [50] found a link between high indigestible protein and rumen undegradable protein (RUP), indicating that low total nitrogen excretion promotes nitrogen retention in the body. YFPKCP: yeast fermented palm cake kernel; SBM: soybean meal; SEM: standard error of the mean (n = 5); L: linear; Q: quadratic; NS: non-significant (p > 0.05); *: significantly different (p < 0.05); **: p < 0.01; a, b, c : Within rows not sharing a common superscript are significantly different (p < 0.05).
All goat groups had a positive nitrogen balance and nitrogen utilization, probably due to receiving a higher nitrogen intake than required. This is most likely owing to NH 3 -N concentrations in the feed exceeding the suggested values for microbial growth and protein synthesis (5-8 mg/dL; Satter and Slyter, [32] or 3.3-8.5 mg/100 mL; Kang-Meznarich and Broderick, [51]). Animals can be kept alive by eating varying levels of YFPKCP as an energy and protein source. Furthermore, as the animal consumes less nitrogen in its diet, it will increase nitrogen retention and decrease nitrogen excretion in order to maintain