Effect of water-washed neem ( Azadirachta indica ) fruit on rumen digesta fatty acids and biohydrogenation intermediates of fattened West African dwarf rams

— This study was conducted to determine the rumen digesta fatty acid profile and biohydrogenation intermediates of West African dwarf rams fattened with diets containing water-washed neem (Azadirachta indica) fruit (WNF). Twenty-five yearling rams (12.3±2.0 kg) were assigned to one of the five dietary groups with five animals per group in a completely randomised design. Each group received a total mixed ration formulated with 0% (T1), 2.5% (T2), 5.0% (T3), 7.5% (T4), and 10.0% (T5) WNF inclusion for 90 days. Chemical analysis was carried out on WNF and the feed using standard procedures. Digesta was collected from the rumen after slaughtering the animals for the determination of fatty acids profile and biohydrogenation intermediates, after the feeding trial. Oleic, palmitic, stearic, and linoleic acids were not significantly (P>0.05) affected by the different treatments. Rumenic acid was linearly lower (P=0.006) in T1 compared to other treatments. The ratio of vaccenic to rumenic acids was linearly and quadratically reduced (P=<0.001) with increased inclusion of WNF. Inclusion of WNF linearly and quadratically increased (P=<0.001) DI-Rumenic acid. The PUFA/SFA was quadratically (P=0.012) lower in T1 compared to T3. Inclusion of water-washed neem fruit in diets of rams increased the concentration of rumenicacid, a conjugated linoleic acid which when incorporated into the animal’s tissue has health promoting benefits when consumed by man. Therefore, the proportion of rumenic acid in mutton should be boosted for increased incorporation into ruminant’s tissues.


INTRODUCTION
The type, composition and activity of rumen microbes are of immense significance to the output of fermentation. This is key to the different strategies for better ruminal function especially, where most often, the output is a product of inefficiently used feed resources. Saturation of fatty acids in the rumen is one of the direct roles played by rumen microbes in an effort to detoxify the rumen ecosystem of excess hydrogen ion for their survival via biohydogenation. Therefore, the output of fat used in ruminant feeding could be improved upon through a proactive approach such as direct microbial interference that is based on an easy to adopt technology.
International Journal of Environment, Agriculture and Biotechnology, 5 (5) Sep-Oct, 2020 | Available: https://ijeab.com/ ISSN: 2456-1878 https://dx.doi.org/10.22161/ijeab.55. 20 1330 The rumen is the habitat for a host of microbes which relate diversely as well as influence fermentative output in a variety of ways. Gram positive or negative bacteria can be suppressed or proliferated by feed constituents and this could either limit or enhance the activities of these microbes on fat alteration in the rumen leading to complete or incomplete metabolism with the production of intermediates. The selective elimination of gram positive bacteria by herbs including neem leaf and enhancement of the gram negatives (Faniyi, 2016) could result in better microbial output due to competitive advantage. Broudiscou et al. (2002) reported the enhancement of some microorganisms by some plant species concomitantly decreased methane production. Reduced methane generation increased conjugated fatty acid production due reduced protozoa or gram positive bacteria which limited the production and availability of hydrogen for biohydrogenation (Ramos Morales et al., 2012).
Neem (Azadirachta indica) fruit is medicinal and could be safely incorporated in ruminants' diets. Neem contains numerous phytochemicals not limited to margosate, nimbidin, nimbin, salanin tannin, saponin, flavonoid, alkaloid, glycoside, and terpenoids, at different concentrations in different plant parts but with proven antimicrobial properties. These phytochemical constituents when beyond the beneficial threshold limit its use. Waterwashing is one of the processing methods for increasing the acceptability and utilization of neem. The ability of neem leaf to increase the total number of beneficial microbes and digestibility in sheep (Faniyi, 2016;Faniyi, 2019) and the fruit to compete favourably with salinomycin in the reduction of coccidial egg count (Tipu et al., 2002;Jack et al., 2020) with reduced methane generation at 5% optimal inclusion level (Jack, 2019) could play a significant role in increasing the efficiency of rumen fermentation and fatty acid transformation for better output. Therefore, rumen digesta fatty acid profile and biohydrogenation intermediates of West African dwarf rams fattened with diets containing waterwashed neem fruit were investigated.

Experimental site
The

Experimental animals, management, and diets
Twenty-five healthy WAD rams (10-12 months) were divided into five groups of five animals in a randomised complete randomised design and housed in well-ventilated and concrete-floored pens containing feeders and waterers. Rams in each group were offered one of the five diets ( Table  1). The experimental diets and potable water were offered ad libitum at 0800 daily for 90 days. Prior to the commencement of the experiment diets, rams were offered the diet with 0% water-washed neem fruit inclusion and administered prophylactic treatments using Ivomec Super, Oxytetracycline (long acting) antibiotics, multivitamins and minerals and allowed a few weeks to acclimatise.  respective rumen digesta sample collected into sterile containers and chilled until used for fatty acid analysis.

Chemical analyses
Proximate analysis, fibre fractions and phytochemical constituents of water-washed neem fruit and feed samples were done as reported in Jack (2019). Fatty acid profile was carried out according to AOCS (1978). Rumenic and vaccenic acids were determined using spectrophotometric method as described by AOAC (2005)

Statistical analysis
Data obtained were analysed using Analysis of Variance (ANOVA) and General Linear Model procedure of SPSS (2006).

Fatty acid intermediates and desaturase index of digesta
Lower rumenic acids (P=0.006) was obtained in T1 compared to other treatments (Table 3). However, increased inclusion of WNF linearly and quadratically reduced VA/RA. Using contrast to compare the means, difference existed for RA except in contrast 5 and 10 while only contrast 10 was not affected in VA/RA (Table 3b). DI-16, DI-18, and total DI were not affected by the different contrast while DI-RA was affected by all the tested contrast. The increase in rumenic acid could be attributed to decreased microbial population and activities, and probably, increased desaturation of vaccenic acids. Increased rumenic acid could by implication be attributed to the reduction in the population of gram positive bacteria which probably could have resulted in the incomplete conversion of fatty acids that are unsaturated to stearic acid thus causing accumulation of intermediate products. Desaturation of precursors such as vaccenic acid could have played a major role as well since bacteria and fungi can undertake desaturation but not protozoa. Devillard et al. (2006) reported the inability of desaturation to occur in protozoal fraction of ruminal digesta.   Rumen biohydrogenation is initially carried out by grampositive bacteria and fungi and subsequently by gramnegative bacteria resulting in the isomerisation of linoleic acid to rumenic acid and the biohydrogenation of rumenic acid to vaccenic acid, and vaccenic acid to stearic acid, respectively Harfoot and Hazzlewood, 1997;Nam and Garnsworthy, 2007). For linolenic acid, after isomerisation, it is followed by progressive hydrogenation to vaccenic acid and then to stearic acid . Therefore, the ratio of RA/LA, VA/RA and SA/VA are indicative of the rate at which the process of ruminal biohydrogenation took place. Higher RA/LA indicated that isomerisation of linoleic acid to rumenic acid was favoured with increased inclusion of waterwashed neem fruit in the diets of rams. Lower VA/RA ratio pointed to the hindrance posed by incorporation of waterwashed neem fruit with respect to the conversion of rumenic acid to vaccenic acid. This could be attributed to reduced microbial activities of Group A bacteria and fungi responsible for the transformation of rumenic acid to vaccenic acid. Desaturation of unsaturated fatty acids in ruminal digesta by bacteria but not protozoa was reported by Devillard et al. (2006) and the ability of fungi (Piromyces communis) to desaturate stearic acid to oleic acid by . DI-rumenic increased with increased inclusion of neem fruit. This may be attributed to increased activity of delta 9-desaturase by bacteria and fungi in the microbial mass.

Estimated fatty characteristics of rumen digesta
Fatty acids as influenced by the different degrees of saturation were not affected by the treatments. However, PUFA/SFA was quadratically lower (P=0.012) in T1 compared to T3 (Table 4). Contrast was not affected in SFA, UFA, MUFA, PUFA, UFA/SFA. However, PUFA/SFA was affected by contrast 1, 2, 6, and 11. Lower PUFA/SFA observed in the control indicated the increase production of SFA over PUFA. This could be as a result of the activities of increased protozoa and bacteria. The hydrogen producing activity of protozoa, fibrolytic fungi and bacteria, particularly gram positive bacteria in the rumen could have made reducing power available for the conversion of unsaturated fatty acids to vaccenic acid. This by implication means that there would have been no formation of stearic acid without the availability of vaccenic acid as provided mainly by the activities of gram positive bacteria. Long chain fatty acids (C18) have antimicrobial effect on bacteria which increased with increase extent of unsaturation with the cis-isomers being to a greater extent disruptive compared to transisomers (Chalupa et al., 1984) and this is in addition to the antimicrobial properties of the phytochemicals in neem fruit. The disruptive effect of these amphiphilic lipids are less on bacteria than on rumen protozoa and fungi (Ushida et al., 1992). Medium chain fatty acids have also been reported to defaunate the rumen in vivo (Machüller and Kreuzer, 1999) and lowered the population of methanogens in vitro (Jordan et al., 2006). These microbes have varying fatty acids composition which on destruction may probably form part of the biomass and would influence the fatty acid composition of the digesta. *Contrast values of P<0.05 are significantly different; 1(WN0 Vs WN2.5, 5, 7.5, 10); 2(WN0 Vs WN5, 7.5, 10); 3(WN0 Vs WN7.5, 10); 4(WN0 Vs WN10); 5(WN0 Vs WN2.5); 6(WN0 Vs WN5); 7(WN0 Vs WN5,10); 8(WN0 Vs 2.5, 5, 10); 9(WN0 Vs WN7.5); 10(WN2.5, 5 Vs WN7.5,10); 11(WN0 Vs WN2.5, 5); 12(WN0 Vs WN5,10).

IV. CONCLUSION
Increased inclusion of water-washed neem fruit increased the concentration of rumenic acid in the digesta. However, further inclusion beyond 5% becomes unnecessary since the concentration of rumenic acid was similar. The increased concentration in digesta if passed down to ruminant tissues will be beneficial to humans when consumed. Rumenic acid is a conjugated fatty acid known for its health promoting benefits. Therefore, increasing the proportion of rumenic acid in mutton will go a long way in promoting health when made available to the tissues.

FUNDING
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.