Determination of In vitro Nutrient Digestibility of Silage based Diets with Varying Levels of Crude Fibre using Pig Faecal Inoculum

.


INTRODUCTION
The use of fibre rich feed ingredients in pig diets especially in small scale farming is rising in recent times due to their wide availability and relatively low cost as compared to commercial feeds. Similarly, offering silages of forages and vegetables in the diets of pigs is becoming an efficient practice in organic pig production system (Edwards, 2002;Bikker et al., 2014;Wüstholz et al., 2017 andFriman et al., 2021). High fibre diets increase cellulolytic bacteria in the large intestines of pigs (Anugwa et al., 1989) and have prebiotic effects and improve gut-associated immune system (Lindberg, 2014). The fermentation products of crude fibre at large intestine such as volatile fatty acids contribute 7 to 25% of maintenance energy needs of pigs (Yen et al., 1991) and butyric acid acts as instant energy source for the colonocytes and regulates cell growth and differentiation (Roediger, 1982). As a part of plant cell wall, crude fibre is generally less digestible in pigs and it adversely effects nutrient utilization (Zhang et al., 2013;Wilfart et al., 2007). The impact of fibre level on nutrients digestibility may vary with fibre source, properties and composition of fibre (Hogberg and Lindberg, 2004;Van Soest, 1978).
Hence, it is imperative to understand the effect of fibre level on nutrient digestibility and voluntary feed intake for optimum swine production. Numerous studies by different scientists regarding digestibility of fibre and fibrous feeds in pigs have been executed. However, information about nutrient digestibility and fermentation characteristics of fibre Determination of digestibility of nutrients is an important criterion for quality evaluation and feeding value. Digestibility evaluation using animal is a time-consuming and tedious process and requires large quantities of feeds and animals. In pigs, digestibility of nutrients starts in the stomach (pepsin and hydrochloric acid), continues in small intestine (enzymatic hydrolysis) and finally in caecum and colon fermentation occurs and help in the breakdown of fibre and other nutrients that had escaped enzymatic hydrolysis. The in vitro nutrient digestibility and fermentation that mimics gastrointestinal process of pigs are convenient methods for rapid and inexpensive measurement of the degradation of feed ingredients (Boisen and Fernandez, 1997;Guo et al., 2004). There may be numerous modifications, where some may use commercially available multi-enzymes (Boisen and Fernandez, 1997) or microbial inoculum either rumen or faecal inoculum (Dung et al., 2002;Bindelle et al., 2007;Youssef and Kamphues, 2020) for simulation of large intestinal fermentation processes. Therefore, vegetable waste silage and maize silage were incorporated as a source of fibre to increase the CF level in experimental diets and were subjected to in-vitro digestion comprising two steps enzymatic hydrolysis and fermentation to investigate the higher level of incorporation of CF in pig feeds.

MATERIALS AND METHODS
The study was conducted at Indian Council of Agricultural Research-National Research Centre on Pig, Rani, Guwahati, Assam, India (9117 49 E longitude, 2548 30 N latitude and 467m altitude) during the month of April-September, 2021. The experiment was carried out taking prior approval of the Institutional Animal Ethics Committee (IAEC) of Indian Council of Agricultural Research-National Research Centre on Pig, Rani, Guwahati and ensured that no potential harm toward animal welfare would be done.
Silages were prepared from maize fodder (Zea mays) and vegetable waste (cabbage and cauliflower at the ratio 60:40 in raw basis) individually in silage bags following standard procedure (3 kg jaggery and 250 g salt per 100 kg raw materials) and opened for sampling after 4 weeks. Feed ingredients were analysed for proximate principles (AOAC, 2012) and neutral detergent soluble and fibre fractions as per the procedure of Van Soest et al. (1991). Energy content of feeds was calculated from the proximate composition using the formula as mentioned by NRC (2012). The pH of silages was estimated following the method described by Bernardes et al. (2019) using digital pH meter (pHTestr 10, Eutech Instruments). The silage quality was determined by using Flieg's score formula (Kilic, 2010). All feed ingredients were dried in oven following standard procedure and ground through a 1mm mesh screen with Wiley No. 4 Laboratory Mill. Low temperature (60C) oven drying method was followed for estimation of dry matter (DM) in silages.

Determination of In vitro Nutrient Digestibility of Silage based Diets with Varying Levels of Crude Fibre using Pig Faecal Inoculum
Representative samples from formulated diets were subjected to a 2-step in-vitro enzymatic hydrolysis to mimic the gastro-intestinal digestion using pepsin and pancreatin solutions (Boisen and Fernández, 1997) followed by in-vitro fermentation as per the procedure described by Youssef and Kamphues (2020) using faecal materials from pigs slaughtered in the institute slaughter house of ICAR-NRC on Pig, Rani, Guwahati.
In-vitro total tract digestibility of DM, CP, OM and CF were obtained after enzymatic hydrolysis and in-vitro fermentation. Cumulative gas production (ml/0.5 g feed) during in-vitro fermentation of experimental diets was also estimated. The data generated were subjected to one way ANOVA as per the methods described by Snedecor and Cochran (1989).

Silage parameters
The prepared silages were evaluated for moisture, pH, fleig point, colour, odour and mould growth ( Table 2). The average moisture content and pH level of vegetable waste silage (VS) was more (75.35%; 4.9) compared to maize silage (MS, 69.35%; 4.0). The moisture content in silage greatly influenced the pH level. Yahaya et al. (2002) opined that at 65-75% moisture level in silage the pH remained below 5; whereas moisture content below or above this value caused increase the pH of orchard grass silages. The average fleig point for maize silage (MS) and vegetable waste silage (VS) was 106 and 59.3, respectively and as per fleig score maize silage was excellent and vegetable silage was fair in quality (Kilic, 2010). VS contained comparatively more CP and energy (18.25%; ME 2536 kcal/kg) than MS (CP 8.3%, ME 2000.8 kcal/kg). Inversely, MS was richer in CF (24.35 vs18.5) and NDF (45.5 vs 28.5) content than VS. Literature pertaining to the chemical composition of vegetable waste silage is scanty to relate the observed findings and it differs as per the content and type of vegetable waste used for silage preparation. The chemical composition of MS was comparable to the report of NDDB (2012) and Htet et al. (2016).

In-vitro dry matter digestibility in enzymatic hydrolysis (IVDMD h )
Diet B1 (62.57%) had significantly higher (P<0.001) IVDMD h ; whereas M3 and V3 diets had significantly lower (P<0.001) IVDMD h as compared to other diets (Table 3). The IVDMD h of diets with normal fibre source was found to be significantly higher (50.80%; P<0.001) as compared to MS or VS containing diets. The CF levels irrespective of fibre sources had significant influence on IVDMD h as shown in Table 4 and digestibility was significantly (P<0.001) reduced as CF level increased in the diet. It may be due to negligible action of pepsin and pancreatin enzymes on fibre and fibre bound nutrients in upper digestive tract.

In-vitro total tract digestibility (IVTTD) of different nutrients
The IVTTD of DM of different diets ranged from 60.80 to 76.54%, where B1 diet showed a significantly higher value (P<0.001) than the other diets. The digestibility of B2 diet was significantly higher (P<0.001) than the M2, M3, V2 and V3 but it was non-significant to M1 and V1 diets. The IVTTD of CP and OM were also significantly higher (P<0.001) for B1 diet compared to other diets, which showed similar trend as the digestibility of DM. The IVTTD of CF was higher in V3 diet (25.85%) and lowest (P<0.001) in M1 diet (11.65%) and showed a highly significant difference to B1, M1, M2, M3, V1 and V2 diets. The higher CF digestibility of high fibre diets may be due to higher concentration of CF as substrate for microbial fermentation (Anguita et al., 2006). The DM, CP and OM digestibility were significantly higher (P<0.001) in the diets that contained normal fibre sources. The CF digestibility of diet containing MS was significantly lower (P<0.01) than the diet with normal feed or vegetable silage as fibre sources. This could be due the quality of fibre and degree of lignification. The diet containing 6% CF level showed a significantly higher (P<0.001) IVTTD of DM and OM followed by 8% CF level; while comparable in diets with 10 and 12% CF level (Table 3). A highly significant difference was recorded for CP digestibility (P<0.001) among the diets with different CF level and diet with 6% CF level showed higher CP digestibility. As the level of CF increased in the diets the IVTTD of CP significantly decreased. On the other hand, the IVTTD of CF was comparable among the diets with different levels of CF (P>0.05). The result obtained is comparable with the outcomes from previous experiment (Gürsoy et al., 2021). Youssef and Kamphues (2020) also reported similar findings when fibre rich ingredients were exposed to in vitro fermentation with faecal inoculum of swine

Cumulative gas production during fermentation
Cumulative gas production at 24 h of in-vitro fermentation ranged from 16.00 to 34.95 (ml/0.5 g feed) and gas production in B2 diet was significantly higher (P<0.001) followed by B1 diet (Table 4). Cumulative gas production of diets with normal fibre source was significantly higher (P<0.001) than MS or VS added diets. It was found that increase fibre level in diet reduces the gas production ( Table 4). The higher production of gas in B1 and B2 diets may be due to higher content of fermentable nutrients and easily digestible fibre. However, cumulative gas production (ml/0.5g feed) in in-vitro fermentation was less compared to experiments of Youssef and Kamphues, (2020) who used substrate without enzymatic hydrolysis; but results were comparable to that reported by Bachmann et al. (2021) for fibre rich ingredients like sugar beet pulp, soybean shell, lucerne and wheat bran after pre-digestion with enzymes. This may be due to the fact that predigested substrate mostly produces less gas, NH 3 and short chain fatty acids as compared to substrate without pre-digestion (Bachmann et al., 2021).

CONCLUSION
The present study revealed that the level and sources of crude fibre in diets influence the digestibility of nutrients. The results obtained from this experiment confirmed the possibilities to use maize silage and vegetable waste silage as CF source in diets of growing-finishing pigs. It is suggested that for better understanding of the role of vegetable waste silage and maize silage as fibre source and their level of incorporation in diets for better health and production needs extensive in-vivo studies in pigs.