Isoquinoline Alkaloids and the Ionophore Monensin Supplemented Alone or Combined on Ruminal Fermentation and Nutrient Digestibility in Steers Fed a High-Energy Diet

Facultad de Medicina Veterinaria y Zootecnia. Universidad Autónoma de Sinaloa. Blvd. San Ángel s/n, Fraccionamiento San Benito CP 80246, Culiacán, Sinaloa, México Instituto de Investigaciones en Ciencias Veterinarias. Universidad Autónoma de Baja California. Km 4.5 carretera Mexicali-San Felipe, CP 21386, Mexicali, Baja California, México Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Col. CU, Coyoacán, CP 04510, Cd. de México, México Department of Animal Science, University of California, Davis, United States Departamento de Ciencias Naturales y Exactas, Universidad Autónoma de Occidente, Unidad Regional Guasave, Avenida Universidad s/n, Flamingos, CP 81048, Guasave, Sinaloa


Introduction
The ionophore Monensin (MON) is commonly included in feedlot cattle growing-finishing diets to enhance feed efficiency. The enhancement in feed efficiency has been attributed, at least in part, to shifts in ruminal fermentation patterns favoring increased propionate and decreased molar proportions of acetate and butyrate (Russell et al., 1988). Although MON may also lead to reduced ruminal microbial efficiency, it may increase flow of non-ammonia N to the small intestine by reducing the ruminal feed protein degradation (Zinn, 1988). Nevertheless, 203 the magnitude of the response to supplemental MON on feed efficiency quite varied, fluctuating from nil (Zinn and Borquez, 1993;Depenbusch et al., 2008) up to increases of 18% (Bartley et al., 1979). A major factor influencing the feed efficiency response to MON is diet energy density (Barreras et al., 2013). Duffield et al. (2012), observed that the magnitude of response of MON on feed efficiency decreased from 8.1 to 3.5% during the past 4 decades, coincident with increases in diet energy density brought by decreased dietary forage levels, increased use of supplemental fat and flaking grain (Samuelson et al., 2016). These changes in diet formulation lend to enhanced ruminal molar proportions of propionate and decreased methanogenesis (Wang et al., 2018). Some phytogenic compounds, such as certain isoquinoline alkaloids (IQA, specifically quaternarybenzo (c) phenanthridine alkaloids sanguinarine and chelerythrine) have effects on ruminal fermentation that could be complementary to MON in high-energy diets. When IQA was added to a substrate comprised of 50:50 forage-to-concentrate, the in vitro molar proportion of acetate increased (Smink and van der Kolk, 2004). Similarly, supplementation of a high-energy diet with a standardized source of IQA (equivalent from 15.8 to 23.8 mg IQA/kg diet DM) increased ruminal acetate molar ratio, but decreasing butyrate in cannulated steers without affect ruminal propionate ratio (Aguilar-Hernández et al., 2016). These same researchers reported that supplemental IQA increased flow of non-ammonia N to small intestine due to both reduced ruminal on feed protein degradation and increased net microbial protein synthesis. These findings indicate a possible synergistic action on digestion and ruminal fermentation in cattle fed with high-energy diets supplemented with both additives combined.
For this reason, the aim of this experiment was to evaluate the effects of supplementation of isoquinoline alkaloids and MON on ruminal fermentation and nutrient digestibility in steers fed a high-energy finishing diet.

Materials and Methods
The trial was conducted at the Ruminant Metabolism Experimental Unit of the Instituto de Investigaciones en Ciencias Veterinarias of the Universidad Autónoma de Baja California located 10 km south of Mexicali City in northwestern México (32° 40' 7" N and 115° 28' 6"W). The area is about 10 m above sea level. All procedures were conducted within approved locally animal care guidelines (NOM, 1999).

Animals, Diets and Sampling
Four Holstein steers [302±15 kg initial shrunk Live Weight (LW)] were fitted with a 3.8 cm i.d. ruminal Tygon "T" cannula and a 1.9 cm i.d. Tygon "T" duodenal cannula (situated approximately 6-cm from pyloric sphincter) with the aim to examine the effects of feeding a combination of Isoquinoline Alkaloids (IQA) and Monensin sodium (MON) in finishing diets on the characteristics of ruminal fermentation and digestive function. Steers were housed in an indoor facility in individual pens (3.9 m 2 ), with a concrete floor covered by a neoprene carpet, automatic waterers and individual feed bunks. Chromic oxide was used as an indigestible marker to estimate nutrient flow and digestibility. Chromic oxide (3.5 g/kg of diet air-dry basis) was premixed with minor ingredients (MON, urea and mineral supplement) in a 2.5 m 3 capacity concrete mixer (mod 30910-7, Coyoacán, Mexico) for 5 min and then, the final product was incorporated after that steam-flaked corn was added to the mixer. Ingredient composition, chemical analysis and calculated dietary net energy (NASEM, 2016) of the basal diet are shown in Table 1. All steers received ad libitum access to the basal diet (Control) for 3 wk before the initiation of the experiment. To avoid feed refusals during experimental period, daily feed intake (as feed basis) was restricted to 90% of observed ad libitum intake during last 7-d of preliminary period (6.9 kg as-fed basis, equivalent to 2.28% of average shrunk initial LW). Treatments consisted of a steam-flaked corn-based finishing diet supplemented as follows: (1) Basal diet with no additives (Control), (2) basal diet plus 15 mg IQA/kg diet DM, (3) basal diet plus 30 mg MON/kg diet DM and (4) basal diet plus 15 mg IQA and 30 mg MON (IQA + MON)/kg diet DM. Source of IQA was Sangrovit RS (Phyto biotics; Futtermittelzusatzstoffe GmbH, Eltville, Germany) containing a standardized mixture of isoquinoline alkaloids, specifically quaternary-benzo (c)-phenanthridine alkaloids, sanguinarine and chelerythrine in a 2:1 ratio with a concentration of 2.25% (w/w). The source of MON was Rumensin 90 (Elanco Animal Health, Greenfield, IN) containing 200 g MON/kg of product. The daily dosage of additives was weighed using a precision balance (Ohaus, mod AS612, Pine Brook, NJ). Supplemental IQA was added (top-dressed) in equal proportions (2.3 g of product per serving) to the basal diet at time the morning and evening feeding, while supplemental Rumensin 90 was premixed with minor ingredients (Cr2O3, urea and mineral supplement) before incorporation into complete mixed diets. The amount of feed of each steer was weighed on a digital scale (Ohaus, NVT 16000/1, México City, México). Diets were fed in two equal proportions at 0800 and 2000 h daily. Experimental periods consisted of 21 days, with 10 days for dietary treatment adjustment, 4 days for collection and 7 days of additive withdrawal (during this period all steers received the control diet). During the collection period, duodenal and fecal samples were taken from all steers following procedure described by Aguilar-Hernández et al. (2016). Briefly, samples were taken twice daily as follows: d 1, 0750 and 1350 h; d 2, 0900 and 1500 h; d 3, 1050 and 1650 h; and d 4, 1200 and 1800 h.

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Individual samples consisted of approximately 500 mL of duodenal chyme and 200 g (wet basis) of fecal material. Samples from each steer and within each collection period were prepared for analysis. During the final day of each period, ruminal samples were obtained to measure microbial populations (protozoa, cellulolytic bacteria and total bacterial). Ruminal samples were prepared and stored by the method described by Dehority (1984) and by Mendoza et al. (1993). During the final day of each collection period, ruminal fluid was obtained, via the ruminal cannula, from each steer at 4 h after feeding. Ruminal sample was taken from the ruminal ventral sac by vacuum pump (Cole Parmer Instrument, Vernon Hill, IL) using a tygon tube (1.9 cm i.d.; USP Lima, Ohio). Ruminal fluid pH was determined on fresh samples. Samples were then strained through four layers of cheese cloth. For VFA analysis, 2 mL of freshly prepared 25% (w/vol) meta-phosphoric acid was added to 8 mL of strained ruminal fluid, centrifuged (17,000 × g for 10 min) and supernatant fluid stored at -20°C. Upon completion of the trial, ruminal fluid was obtained from all steers and composited for isolation of ruminal bacteria via differential centrifugation (Bergen et al., 1968) as follows: (1) ruminal fluid was diluted 50:50 with 0.16N saline (37°C) agitate gently for about 30 seconds and strained through 4 layers of cheesecloth; (2) strained fluid was promptly transferred into centrifuge bottles and spun at 2000 × g for 10 min at 10°C; (3) supernate was decanted and centrifuged at 43,000 × g for 20 min at 10°C and (4) supernate was decanted and the pellet isolated, oven-dried (70°C) and then ground with a mortar and pestle. The microbial isolate served as the purine: N reference for the estimation of microbial N contribution to chyme entering the small intestine (Zinn and Owens, 1986). Additionally, during the final day of each period, blood samples (5 mL) were taken via jugular vein 5 h post-feeding in order to determine the enzymes Gamma-Glutamyl Transferase (GGT) and aspartate Aminotransferase (AST), analyzed enzymatically on a Beckman Olympus AU640 auto analyzer (Myko Analytical, Lake Tapps, WA, USA). These enzymes were measured as indicators of possible liver damage due to supplemental additives.

Sample Analysis and Calculations
Feed, duodenal and fecal samples were subject to the following analysis: Dry matter (method 930.15); ash (method 942.05) and Kjeldahl N (method 984.13) following the procedures published by AOAC (2000). Neutral detergent fiber [aNDFom, corrected for NDF-ash, incorporating heat stable α-amylase (Ankom Technology, Macedon, NY) at 1 mL per 100 mL of NDF solution (Midland Scientific, Omaha, NE)] was determined following the procedures described by Van Soest et al. (1991) and chromic oxide (Hill and Anderson, 1958) and starch (Zinn, 1990). In addition, ammonia-N (method 941.04; (AOAC, 2000) and purines (Zinn and Owens, 1986) were determined in duodenal samples. Concentrations of VFA in ruminal fluid were determined by gas chromatography (Zinn, 1988). The counting procedures for total protozoa and total bacterial were performed according to Dehority et al. (1989). Cellulolytic bacteria was cultured and counted by the method described by Van Gylswyk and Hoffman (1970). Bacterial and protozoal counts are expressed as log10/mL.
Total DM flow to the duodenum and fecal excretion were estimated using Cr2O3 as an external marker. Feed, duodenal and fecal OM was determined by difference between DM and ash content. Microbial Organic Matter (MOM) and Microbial Nitrogen (MN) leaving the abomasum (as obtained from a duodenal cannula placed approximately 6 cm from the pyloric sphincter) were calculated using purines as a microbial marker (Zinn and Owens, 1986). Organic matter truly fermented in the rumen was considered equal to the OM intake minus the difference between the amount of total OM reaching the duodenum and the MOM reaching the duodenum. Feed N escape to the small intestine is considered equal to the total N leaving the abomasum minus the sum of ammonia-N plus MN reaching duodenum and, thus, includes any endogenous N contributions. Ruminal microbial efficiency was estimated as duodenal MN, g /kg OM fermented in the rumen and protein efficiency represent the duodenal non-ammonia-N, g/g N intake.

Statistical Design and Analysis
Treatment effects on characteristics of digestion were analyzed as a balanced 4 × 4 Latin square design in a 2 × 2 factorial arrangements using the MIXED procedure according to SAS (2004). The fixed effect consisted of treatment and random effects consisted of steer and period. The statistical model for the trial was as follows: where: Yijk is the response variable, µ is the common experimental effect, Si is the steer effect, Pj is the period effect, Tk is the treatment effect and Eijk is the residual error. Treatment effects were separated into the following orthogonal contrasts: (1) Non-additive vs. IQA; (2) non additive vs MON; and (3) IQA× MON interaction. In addition, means separations were performed using Fisher's LSD. Contrasts are considered significant when the P value was ≤0.05 and as tendencies when the P-value was >0.05 and ≤0.10.

Results and Discussion
Treatment effects on characteristics of ruminal and total tract digestion are summarized in Table 2. Flow of ammonia-N to the small intestine was greater (interaction, P<0.01) for non-supplemented control than for the other three treatments. Flow non-ammonia N (NAN; interaction, P = 0.04) was greater for supplemental IQA alone than for the other three treatments. When IQA was added to the control diet, duodenal flow of NAN increased (9.2%) and NH3-N flow decreased (23.7%). In contrast, when IQA and MON were added to the Control diet no effect on NAN flow to the small intestine was observed and NH3-N flow decreased only 18.2%. This interaction was also observed (P<0.05) in protein efficiency (NAN flow to the small intestine/N intake) and postruminal N digestion. Addition of IQA to control diet resulted in a 9.1% increase in protein efficiency and 6.1% increase in postruminal N digestion. Whereas, IQA in combination with MON did not increase (interaction, P<0.05) protein efficiency or postruminal N digestion. The basis for this response is unclear. Earlier reports indicate that MON decrease ruminal concentration of microorganisms with high proteolytic activity and it may have direct effect on protease and deaminase enzymes as well (Bergen and Bates, 1984;Russell and Strobel, 1989). On the other hand, IQA could modulate metabolism of rumen microbes with minor changes on rumen microbial population and species diversity (Petri et al., 2019). The effects of both additives alone, decrease the ruminal degradation of feed N; however based on the duodenal flows of NAN, ruminal scape feed N and microbial N with the combination, apparently the effects of each additive are negatively affected. More research is is needed to further assess these interactions, as well as possible interactions with other additives (including alternative ionophores) that may be supplemented in feedlot diets.
IQA supplementation increased (5.6%, P = 0.02) flow of NAN and decreased (11.8%, P = 0.01) flow of NH3-N to the small intestine. The increase in NAN flow to the small intestine was due in part to increased (6.5%, P = 0.01) flow of microbial N to the small intestine. This increase is a reflection of increased (7.6%, P = 0.03) ruminal microbial efficiency (expressed as duodenal MN, g/kg OM fermented in the rumen) and increased (9.1%, P = 0.02) ruminal protein efficiency (expressed as duodenal non-ammonia N, g·g-1 N intake). Aguilar-Hernández et al. (2016) observed that in steers fed a diet similar that of the present study, 206 supplementation with 16.8 mg IQA/kg diet DM decreased ruminal ammonia N concentration; presumably due to decreased proteolysis and deamination of amino acids (Drsata et al., 1996). More recently, Petri et al. (2019) likewise observed decreased amino acids metabolism in IQA supplemented treatments. They noted that predicted amino acid metabolism pathways were down-regulated in all IQA supplemented groups in comparison to the control group, a key mode of action for these IQA supplementation in regard to improving ruminal amino acid bypass. The increase of net microbial N flow to duodenum in steers fed IQA may be partially explained (as indicated below) by reduced recycling of microbial protein as consequence of decreased ruminal protozoa.
There Supplemental MON decreased (9.4%, P = 0.03) flow of ammonia-N and NAN (4.8%, P = 0.02) to the small intestine and in turn, ruminal N efficiency (5.7%, P = 0.02). The latter is attributable to decreased (7.4%, P = 0.02) ruminal microbial efficiency and associated decrease (8.6%, P = 0.02) in microbial N flow to the small intestine. Consistent with previous studies (Morris et al., 1990;Salinas-Chavira et al., 2009), supplemental MON did not affect (P>0.10) site and extent of OM, NDF and starch digestion. Comparable effects of supplemental MON on feed N degradation and microbial synthesis in feedlot steers has been reported previously (Zinn, 1987;Zinn et al., 1994). Due to antimicrobial properties of MON some decrease in ruminal NDF degradation can be expected. However, the effects of MON of fiber digestion has not be consistent (Salinas-Chavira et al., 2009). Both increases and decreases in fiber digestion have been associated with ionophore feeding (Spears, 1990). Varying effects are apparently dependent on fiber level and source. As with the present study, NDF levels are low in conventional finishing diets for feedlot. Due to high dietary starch and consequent low ruminal pH, the extent of ruminal fiber digestion is low (≤40%), independently of MON supplementation. Although, in the present study MON numerically decreased (10%, P = 0.18) ruminal digestion of NDF.
Treatment effects on ruminal pH, VFA molar proportions are shown in Table 3. There were no treatment main effects, or synergism (P>0.10), on ruminal pH. Contrary to our hypothesis, there was no treatment synergism (P>0.10), on ruminal VFA proportions. However, IQA increased (10.9%, P = 0.02) acetate molar ratio, this effect reflected a tendency of increase (P = 0.06) the acetate: Propionate molar ratio. The absence of effects of IQA on ruminal pH and total VFA production are consistent with lack of treatment effect on ruminal OM digestion. Similar findings have been reported for steers fed both medium energy diets (Petri et al., 2019) and high energy diets (Aguilar-Hernández et al., 2016;Zhang et al., 2019). In vitro studies of Smink and van der Kolk (2004) also showed increased acetate: Propionate molar ratio without effect on total VFA production. However, Khiaosa-ard et al. (2020) using a rumen simulation technique (Rusitec) noted that IQA supplementation increased ruminal propionate at dose of 8.75 mg IQA/kg DM, but at dose of 17.50 mg IQA/kg DM did not registered differences between ruminal molar proportion of VFA in a medium-energy substrate (35:55 forage concentrate ratio).
Lack of an influence of MON on ruminal pH, VFA molar proportions is consistent with numerous studies in which MON was supplemented in high-energy diets (Zinn et al., 1994;Salinas-Chavira et al., 2009;Felix and Loerch, 2011).
Treatments effects on ruminal microbial counts are shown in Table 4. Combining IQA with MON increased (interaction, P < 0.01) total protozoa count and nullified the effects (interaction P = 0.04) of MON on the ruminal total bacterial count. It has been previously observed that combinations of antimicrobials may act differently on microorganisms than when they are administered separately (Mitosch and Ballenbach, 2014).
There were no treatment effects (P > 0.16) on cellulolytic bacteria counts. The total bacteria count was lower (interaction, P = 0.04) for MON alone than for the other three treatments. Supplemental IQA alone decreased (interaction, P<0.01) ruminal protozoa counts compared to that of controls (28%) and MON, alone (21%). Whereas protozoal counts for control and MON plus IQA were not different. Likewise, Petri et al. (2019) using a rumen simulation technique (Rusitec) noted that IQA supplementation decreased ruminal protozoa population. Sanguinarine and chelerythrine, principal compounds on IQA source used here, have a significant dose-dependent antibacterial activity (since 16 µg/mL) against Gram-positive and Gram-negative bacteria when tested in vitro (Opletal et al., 2014). Protozoal recycling of microbial protein depresses ruminal microbial efficiency (net flow of microbial N to the small intestine). The decrease in ruminal protozoal counts and concomitant increase in observed microbial efficiency with IQA supplementation is consistent with this observation.
Development of MON resistance has been shown to occur in both gram-positive and negative species (Russell and Strobel, 1988). Apparently, prolonged use of MON alters the ruminal microbial ecosystem, selecting for ionophore-resistant members of the microbial population (Callaway et al., 2003). Although MON has consistently reduced in vitro protozoal counts, that effect is less consistently observed in vivo (Russell and Houlihan, 2003).

Conclusion
Isoquinoline Alkaloids mixture (IQA) supplementation improve of N utilization by promotes a lesser ruminal degradation of N and by greater microbial N flow to the small intestine. The inclusion of IQA to the diet increase molar proportion of ruminal acetate. IQA plus MON combination failed to be synergic on digestion nor ruminal fermentation, in opposite, had a negative associative effect on N utilization. More research is needed to further assess these interactions, as well as possible interactions with other additives (including alternative ionophores) that may be supplemented in feedlot diets. Isoquinoline alkaloids mixture (IQA) represent a strategy to improve N utilization in ruminants fed high-energy diets.