Effect of oxidized β-carotene-oxygen copolymer compound on health and performance of pre- and post-weaned pigs

The discovery that a naturally occurring, biologically active β-carotene-oxygen copolymer compound is the main product formed in spontaneously oxidized β-carotene has stimulated interest in its potential health benefits. The copolymer, formed in nature or synthetically by the air-oxidation of β-carotene, possesses beneficial immune modulating activities that previously had been attributed to β-carotene itself. Support for these benefits is provided by previous studies showing that supplementation in feed with low parts-per-million levels of copolymer-rich, fully oxidized β-carotene (OxBC) helped reduce the negative impact of subclinical necrotic enteritis in broilers and improved growth in weaned piglets. To further assess these potential benefits, two trials were conducted in swine raised under commercial conditions in Vietnam. Trial 1, a 140-day full-grow, post-wean study with 500 28-day-old pigs, compared 2, 4 or 8 ppm OxBC against both an unsupplemented and an antibiotic control group. OxBC and antibiotics each improved growth rate, feed efficiency, and body weight compared to the control (P<0.001). Animals receiving 4 and 8 ppm OxBC performed better than did animals on antibiotics (P<0.001). In starter pigs, OxBC reduced the occurrence of diarrhea dose-dependently (4 and 8 ppm) and to a greater extent than did antibiotics (P<0.001). Trial 2, a 49-day study with 420 piglets, was conducted in two-stages. In Stage 1 (pre-wean), OxBC in the transition (creep) feed produced a dose-dependent trend toward increased body weight over 21 days, reaching significance at the highest inclusion level (16 ppm) (P<0.001). In Stage 2 (post-wean), body weight gain showed a dose-dependent trend and was significant for both 8 ppm OxBC and the antibiotics at 28 days post-wean (P<0.001). Feed conversion was better at 8 ppm OxBC and for the antibiotic group (P<0.001). These findings support the concept that β-carotene-oxygen copolymers help optimize immune function, and provide validation for the effectiveness of this strategy in enhancing animal performance in the absence of in-feed antibiotics.

compound is the main product formed in spontaneously oxidized β -carotene has stimulated 19 interest in its potential health benefits. The copolymer, formed in nature or synthetically by 20 the air-oxidation of β -carotene, possesses beneficial immune modulating activities that 21 previously had been attributed to β -carotene itself. Support for these benefits is provided by 22 previous studies showing that supplementation in feed with low parts-per-million levels of 23 copolymer-rich, fully oxidized β -carotene (OxBC) helped reduce the negative impact of 24 subclinical necrotic enteritis in broilers and improved growth in weaned piglets. To further 25 assess these potential benefits, two trials were conducted in swine raised under commercial 26 conditions in Vietnam. Trial 1, a 140-day full-grow, post-wean study with 500 28-day-old 27 pigs, compared 2, 4 or 8 ppm OxBC against both an unsupplemented and an antibiotic 28 control group. OxBC and antibiotics each improved growth rate, feed efficiency, and body 29 weight compared to the control (P<0.001). Animals receiving 4 and 8 ppm OxBC 30 performed better than did animals on antibiotics (P<0.001). In starter pigs, OxBC reduced 31 the occurrence of diarrhea dose-dependently (4 and 8 ppm) and to a greater extent than did 32 antibiotics (P<0.001). Trial 2, a 49-day study with 420 piglets, was conducted in two-stages. After one day post-farrowing, sows with minimum litter sizes of ten piglets and 163 similar weights were selected. Litter sizes were also standardized by cross-fostering 164 between sows within treatment groups within three days post-farrowing. As most sows had 165 litter sizes greater than ten, surplus piglets were cross-fostered to sows that were not part of 166 the trial. 167 168 In Stage 1, 42 lactating sows (one week post-farrow) and their one-week-old 169 offspring were randomly and evenly assigned to one of six treatments with seven replicate 170 pens per treatment. Each pen contained one sow and her litter of ten piglets. The mean 171 piglet body weight was 2.44 kg. As there were insufficient sows to carry out seven 172 replicates simultaneously, the trial was run in a time-replicated fashion, with two cohorts of 173 sows. The first cohort contained 18 sows with 180 piglets (six treatments and three 9 replicates) and the second cohort contained 24 sows with 240 piglets (six treatments and 175 four replicates). The second cohort began the trial one week after the first cohort. 176

Stage 1. Pre-wean (Days 0-21)
The diets were introduced as soon as the piglets were ready to begin consuming 177 creep feed. Sows in all groups received a basal commercial lactation diet without OxBC 178 supplementation. Dietary treatments were delivered to piglets via supplementation of the 179 creep feed as follows: Control (basal diet with no antibiotic or OxBC), AB (basal diet with 180 antibiotics) and OxBC (basal diet with 2, 4, 8 or 16 ppm OxBC; no antibiotic). The 181 composition of the basal diet is shown in Table 2. 182 Feed and water were provided ad libitum. Piglets were individually weighed at the 183 start of the study (Day 1) and at weaning (Day 21). Feed given was weighed daily and 184 unused feed was weighed at the end of each week. 185 186 Stage 2 began at Study Day 22, one day post-wean, when the piglets were 29 days old. 187

Stage 2. Post-wean (Days 22-49)
Three hundred and sixty (360) piglets were used in a random block design of six treatments 188 with six replicate pens of ten piglets each. Piglets were randomized within their prior 189 treatment groups before being equally assigned into pens. Treatments were as follows: College, PA, USA). Differences between means were tested using Fisher's multiple range 207 test when the F value was significant at P< 0.05. Data were considered significantly 208 different at P<0.05. 209

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Trial 1. Full grow study 211 The effect of dietary supplementation with OxBC on the growth performance of pigs 212 is shown in Table 3. On Day 1, there were no significant differences in BWs among any of 213 the groups (P>0.05). Dietary supplementation with all three levels of OxBC during the 214 Starter period (Days 1 to 28) resulted in significant improvements in body weight (BW), 215 average daily weight gain (ADG), average daily feed intake (ADFI) and feed/gain ratio 216 (F/G) compared to the control group (P<0.001). Animals in the OxBC groups also 217 performed better than or equivalent to those receiving antibiotics during the starter period. 218 During the Grower period (Days 29-84), supplementation with all levels of OxBC 219 significantly increased ADG relative to the Control (P<0.001). The ADG of the AB group 220 was also higher than the Control (P<0.001), but there were no differences among the OxBC 221 and the AB groups. There was an apparent dose-dependent decrease in feed consumption 222 across the three OxBC groups, with the ADFI of the 8 ppm OxBC group being significantly 223 lower than the Control (P<0.05). The F/G ratios for all OxBC groups and the AB group 224 were significantly improved compared to the Control (P<0.001). The 8 ppm OxBC group 225 had the lowest F/G ratio, which was significantly lower than the ratios for the Control, AB In terms of clinical health, the results presented in Table 4 show that the highest 243 incidences of both diarrhea and mortality were observed in the Control group for all study 244 periods, with the lowest being observed in the OxBC groups. The highest incidence of 245 diarrhea was during the Starter period, and OxBC significantly and dose-dependently 246 reduced the diarrhea rate (P<0.001). Diarrhea was the lowest (less than half that of the 247 Control group) in the 8 ppm OxBC group, and both the 4 and 8 ppm OxBC groups had 248 significantly reduced incidences relative to the AB group (P<0.001). 249 The occurrence of diarrhea during the Grower and Finisher periods was markedly 250 less compared to the Starter period. Despite the lower background incidence of diarrhea in 251 these later growth phases, OxBC still continued to provide significant and dose-dependent 252 protection from the condition. 253 Overall, animals in the Control group had the highest incidence, whereas animals in 254 the 8 ppm OxBC group had the lowest incidence. OxBC significantly and dose-dependently 255 reduced the incidence relative to both the Control and AB groups (P<0.001). 256 Mortality was relatively low, overall, with few pigs dying during the study. The 257 lowest number of deaths occurred in the OxBC groups, but differences were not significant 258 between any of the groups. 259 Trial 2: Pre-and post-wean piglet study 260 261 The effects of OxBC and antibiotics on the growth performance of pre-weaning 262 piglets are provided in Table 5. 263

Stage 1 Pre-weaning (Days 1-21)
Inclusion of OxBC in the creep feed resulted in a dose-dependent trend towards 264 increased ADG and BW that reached significance for 16 ppm OxBC compared to the 265 Control over the 21 days of Stage 1. The ADG and BW of the AB group were also higher 266 than the Control. Antibiotics and 16 ppm OxBC improved the F/G ratio compared to the 267 13 Control, while the ADFI was not significantly different across the 6 treatment groups 268 (P>0.05). 269 The very low F/G ratio observed for all groups is attributed to the small amounts of 270 feed ingested by the piglets, which were still mainly consuming milk from the sows. 271 14 No evidence of diarrhea or mortality was observed in Stage 1. 272 273 The effects of OxBC and antibiotics on BW, ADG, ADFI and F/G for weaned 274 piglets are shown in Table 6. 275

Trial 2: Stage 2 (Days 22-49)
At the beginning of Stage 2, a subset of select piglets from each treatment group was 276 randomized within treatment groups to continue the trial. Piglets were selected such that the 277 average BW among the six treatment groups did not differ on Day 22 (the beginning of 278 Stage 2). After 28 days (Day 49, the end of Stage 2), average BWs were highest for the AB 279 group followed by the four OxBC groups and the Control. Increases in average BW reached 280 statistical significance for both the AB and the 8 ppm OxBC groups (P<0.001). 281 The increases in BW were reflected in the ADG. The AB group had the highest 282 ADG, which was significantly higher than the Control (P<0.001). ADG showed a trend 283 towards a dose-dependent increase in the OxBC groups, reaching statistical significance for 284 the 8 ppm OxBC group compared to the Control. However, there was no statistical 285 difference in ADG across the four OxBC groups. 286 There were no significant differences in ADFI among the treatment groups. F/G 287 ratios were significantly improved for both the AB group and the 8 ppm OxBC group 288 compared to all other groups (P<0.001). 289 The effects on the incidence of diarrhea and mortality are given in Table 7. Diarrhea 290 and mortality incidence were generally low in all groups, with the highest incidence of 291 diarrhea and mortality being observed in the Control group. Treatment with 8 ppm OxBC or 292 antibiotics decreased diarrhea incidence by 24% and 28%, respectively. 293 No mortalities were recorded in the AB group, the 8 ppm OxBC group or the group 294 fed 16 ppm OxBC in Stage 1 and 4 ppm OxBC in Stage 2. The differences in mortalities 295 between the groups were not significant. 296

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Results from the two trials reported here demonstrate that dietary supplementation 298 with low parts-per-million levels of OxBC improves the health and productivity of pigs 299 reared under commercial production conditions. In Trial 1, conducted over the entire 140-300 day post-wean grow-out period, inclusion of OxBC in the feed led to improvements in 301 growth performance during each of the three stages of the production cycle and also 302 significantly reduced the incidence of diarrhea in post-wean starter piglets. Results from the 303 second trial confirm the benefits of dietary OxBC on growth performance of post-wean 304 starter piglets and indicate that further benefits on piglet performance are possible when 305 OxBC is added to the transition (creep) feed of nursing piglets prior to weaning. 306 In Trial 1, the observation that the benefits of OxBC were most apparent during the 307 starter period compared to the grower and finisher periods is not surprising, given that 308 The improvement in clinical health (reduced incidence of diarrhea) likely explains, 339 in part, the improved growth performance of piglets in the OxBC-supplemented versus 340 control groups during the Starter period in Trial 1. As would be expected for older, more 341 immunocompetent pigs, there were fewer incidences of diarrhea in grower and finisher pigs 342 compared to starter pigs across all treatment groups in the first trial. While there tended to 343 be a lower incidence of diarrhea in the OxBC supplemented groups compared to the control 344 during the grower and finisher periods, it seems unlikely that these small reductions in 345 disease incidence would explain the improved growth performance observed in the OxBC 346 groups during the latter stages of the trial. Instead, the ability of OxBC to reduce 347 inflammatory tone represents the most plausible mechanism to explain the improved 348 growth of older pigs in the OxBC groups. there was no significant treatment effect (Table 7). There were considerably fewer cases of 375 diarrhea observed in the control group in Trial 2 (4.7%) compared to Trial 1 (7.9%). This 376 difference in the background incidence of diarrhea in the two trials likely reflects a lower 377 level of endemic pathogen pressure during Trial 2 relative to Trial 1. With a relatively low 378 incidence of diarrhea observed in the control group in Trial 2, further treatment-induced 379 reductions may not have been possible. Thus, in Trial 2 the benefits of OxBC on growth 380 performance of post-wean piglets is more plausibly due to anti-inflammatory actions rather 381 than a reduction in the incidence of diarrhea. It is interesting to note that the magnitude of 382 the performance benefits in piglets was much larger in Trial 1 compared to Trial 2. The 383 difference in the magnitude of the benefits may have been due to a combined benefit of 384 reducing diarrhea and background inflammation in the gut in Trial 1, while in the second 385 trial, where there was lower endemic pathogen pressure, only the anti-inflammatory activity 386 may have factored in the benefit. 387 The results from the pre-wean period (Stage 1) of Trial 2 indicate that inclusion of 388 OxBC in transition or creep feed can also improve weight gain and body weight of nursing 389 piglets. OxBC appeared to dose-dependently increase ADG and body weight at weaning 390 with the highest inclusion rate of 16 ppm OxBC reaching statistical significance (Table 6).    Stage 1 to account for the low feed intake of nursing piglets. 567