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The effects of using shrimp meal in broiler diets on live performance and carcass traits
Lourdes I. Maldonado and Héctor Santiago
Department of Animal Industry, University of Puerto Rico at Mayagüez
Abstract
An experiment was conducted to evaluate the effect of the inclusion of different levels of shrimp meal
(SM) in diets on growth performance and carcass traits of broilers. A total of 160 d-old chicks were
randomized in four treatments with four replicate pens of 10 birds each. Treatments consisted of SM
inclusion at 0 (Control), 3, 6, and 9% in both starter (0-21 d) and grower (22 - 49 d) diets. Birds were
raised under standard commercial conditions and provided with feed and water ad-libitum. Feed and
birds were weighed weekly up to 49 d to determine bodyweight (BW), feed intake (FI), and feed
conversion (FC). There was a negative linear response (P < .05) in BW with increasing levels of SM in
the diet until 35 d of age. BW decreased as the dietary percentage of SM increased. At all ages, control
birds were heavier (P

Introduction
The broiler industry is the second largest agricultural sector of Puerto Rico. Broiler production
costs here are higher than in other countries due to the fact that there is no local production of
grains and most feed ingredients used in poultry diets are imported. Therefore, to reduce feed
costs it is crucial to find novel feedstuffs that are feasible, available, economical, and of
adequate nutritional value, that can substitute for at least part of the imported ingredients
conventionally used in broiler diets.
A local aquaculture company produces annually some 1,800 tons of salt water shrimp. Since
the heads comprise about 44% of whole raw shrimp (Meyers and Rutledge, 1971) we estimate
that this company alone generates on the order of 800 tons of waste that is sent to land fills for
disposal. Shrimp meal (SM), the dried waste of the shrimp industry, is comprised of heads,
hulls, shrimp not suitable for market, and fish accidentally caught with shrimp during harvest.
SM contains approximately 50% crude protein on a dry matter basis (Rosenfeld et al., 1997 and
Gernat, 2001) and could be a valuable feed ingredient to partially or totally replace soybean
meal in broiler diets. Shrimp meal has been used successfully as a feed ingredient in diets of
laying hens (Rosenfeld et al., 1997) and broilers (Singletary et al, 1935; Ewing, 1963; Damron
et al., 1964; Ilian et al., 1985, and Islam et al., 1994; Gernat, 2001). In these studies the use
of shrimp meal in broiler diets had no detrimental effect on performance and organoleptic
properties of breast meat. However, no studies could be found evaluating the effects of the
use of shrimp meal on other carcass traits.
Materials and Methods
Shrimp wastes were collected from the processing facilities of Eureka Marine Products in
Dorado. Wastes were transferred to an 8’ x 8’ air steam dryer and dried at 65° C for 72 h. The
dried wastes were grounded to a particle size of 1.0 mm. A proximate analysis and amino acid
profile were performed on the final product prior to diet formulation according to methods of
AOAC (1990). A two phase feeding regime consisting of starter (0 to 21 d) and grower (22 to
49 d) was used in the experiment. Diets were corn and soybean meal based and formulated to
meet or exceed the NRC recommendation for broilers (NRC, 1994). The treatments consisted
on inclusion of SM at 0 (control), 3, 6, and 9% of the diet to partially substitute the crude
protein contributed by soybean meal.
2

A total of 160 broiler chicks were raised under standard commercial conditions in an open-sided
poultry house at the Small Animal Research Farm in Lajas of the Department of Animal
Industry. Birds were randomly assigned to 16 floor pens with four replicate pens per treatment.
Chicks were raised under a 24 h of light photo regimen and provided with sufficient feed and
water for ad-libitum consumption. Birds and feed were weighed at 7, 14, 21, 28, 35, 42, and 49
d of age to determine bodyweight (BW), feed intake (FI), and feed conversion (FC).
At 49 d, four birds per replicate pen for a total of 16 per treatment were randomly selected,
wing banded and kept 12 h prior to slaughter without access to feed. After feed withdrawal
birds were weighed, exanguinated, scalded, mechanically plucked, and manually eviscerated.
Carcasses were chilled overnight in an ice-slush tank maintained at 0 °C. After chilling,
carcasses were drained, reweighed, and cut into the following portions: wings, drumsticks,
thighs, breast with ribs, back, and neck. Carcass parts and abdominal fats were weighted and
the yield expressed as percentage of live bodyweight (LBW).
Data were analyzed according to a Completely Randomized design by ANOVA using the GLM
procedures of SAS ® (SAS Institute, 1990). The Tukey multiple comparison test was used to
separate treatment means. All statements of significance are based on a probability of P< 0.05.
Results and Discussion
As shown in Table 1, growth performance of control birds was superior to that of those fed 9%
SM diets. At all ages, control birds were significantly heavier than those fed 9% SM, while BW
of those receiving 3 and 6% SM were intermediate; 3 % SM differed from the control at 7 d
and again at 35 d, while at 42 d both 3 and 6% SM were superior to 9% SM. There was a
significant negative linear response in BW with increasing levels of SM in the diet at all ages.
BW decreased as the percentage of SM increased in the diet. No significant differences in FI
were observed in either the starter or grower periods (Table 1). During the starter period FC
was similar for all treatments averaging 2.16 overall and ranging from 1.94 to 2.33 (g feed/ g
BW). At 49 d, control (2.44) and birds fed 3% SM (2.71) had significantly lower FC than those
fed 9% SM (4.50), while FC of birds fed 6% SM (3.30) did not differ from the other treatments.
Cunha et al. (2003a) found that 0, 3, 9 and 12% of SM inclusion in broiler diets resulted in no
significant differences in FI or FC. Thus there is agreement with results of the present study in
FI, but disagreement in the effects of higher levels of SM inclusion on FC. Those researchers
3

observed a quadratic effect between BW and SM level wherein the best levels of inclusion were
5.46, 5.87, and 6.16%.
Table 1. The effect of SM included at different levels in broiler diets on body weight and feed conversion
0% SM 3% SM 6% SM 9% SM
Age (d) Body weight (g)
1 42 a
42 a
42 a
42 a
7 139 a
131 ab
125 b
125 b
14 320 a
285 ab
296 ab
261 b
21 527 a
479 ab
459 ab
430 b
28 959 a
852 ab
840 ab
761 b
35 1,428 a
1,305 ab
1,261 b
1,061 c
42 1,860 a
1,739 a
1,669 a
1,400 b
49 2,365 a
2,242 ab
2,241 ab
1,941 b
Feed Convertion (g feed / g body weight)
Starter (1 – 21 d) 1.94 a
Grower (22 – 49 d) 2.44 a
2.16 a
2.33 a
2.71 a
3.30 ab
a-b
Means in a given row bearing different superscripts differ significantly at P < 0.05.
2.20 a
4.50 b
The LBW and plucked carcass weight (PCW) of processed birds were similar for control, 3 and
6%, but significantly higher than for those fed 9% SM (Table 2). Control dressed carcass
weight (DCW) was significantly heavier than those of birds fed 6 and 9% SM. LBW, PCW, and
DCW tended to decrease as the percentage inclusion of SM increased in the diet. No
differences in plucked carcass yield were observed among treatments, values ranging from 88.7
to 89.7 %. However, a higher dressed carcass yield was observed for the control birds than for
those of 6 and 9% SM. As shown in Table 3, no significant differences in yields of abdominal
fat, back, neck, wing, and drumsticks were observed among treatments. The breast yield of
control, 3% and 6% fed birds were similar but significantly higher than that of 9% SM birds.
The same was true of thigh yield except that the difference between the control and 9% SM
was not significant. In a similar experiment, Cunha et al. (2003b) reported no significant
differences in breast, thigh, and drumstick yields of broilers feed 0, 3, 6, 9 and 12% SM.
Conclusion
The results of this study indicate that SM shows promise as a potential feed ingredient to supply
part of the dietary protein requirements of broilers, but its inclusion should be limited to
between 3 and 6% of the diet in order not to sacrifice growth performance and desirable
carcass traits.
4

Table 2. The effect of SM included at different levels in broiler diets on live body weight, lucked
weight, hot dressed weight, cold dressed weight and percentage plucked, hot and cold
dressed yield 1, 2 .
Trait 0% SM 3% SM 6% SM 9% SM
Live body weight 3 (g)
Plucked weight 4 (g)
Hot dressed weight 5 (g)
Cold dressed weight 6 (g)
2,548 a
2,285 a
1,771 a
1,887 a
2,365 a
2,179 a
1,639 ab
1,747 ab
2,319 a
2,063 a
1,546 b
1,649 b
1,884 b
1,683 b
1,236 c
1,331 c
Plucked carcass yield (%) 7 89.7 89.5 88.8 88.7
Hot dressed yield 8 (%)
Cold dressed yield 9 (%)
69.5 a
74.0 a
67.1 ab
71.6 ab
66.5 ab
70.9 b
a-b
Means in a given row bearing different superscripts differ significantly at P < 0.05.
1
Values given in this table correspond to least-squares means obtained from ANOVA and their pooled SEM.
2
n = 17 birds per mean
3
Body weight prior to slaughter following feed and water deprivation for 10h
4
Carcass weight after exanguination prior to evisceration with legs and neck left in the carcass.
5
Without giblets but with abdominal fat pad left in the carcass.
6
Without giblets after chilled in an ice-slush tank for 24 h
7
Plucked carcass weight / live BW after feed and water deprivation x 100.
8
Hot carcass weight / live BW after feed and water deprivation x 100.
9
Cold carcass weight / live BW after feed and water deprivation x 100.
Table 3. The effect of SM included at different levels in broiler diets on carcass cuts and
abdominal fat yields as percentage of live weight 1, 2 .
Carcass Cut 0% SM 3% SM 6% SM 9% SM
Neck 4.1 4.4 4.2 4.1
Feet 20.2 b 22.4 a 22.3 a 22.6 a
Back 15.2 13.7 13.7 14.5
Wing 16.7 16.5 15.8 16.9
Thigh 23.4 ab 24.8 a 24.7 a 21.3 b
Drumstick 22.5 22.3 22.4 24.2
Breast 52.2 a 49.3 a 48.2 a 40.3 b
Abdominal fats 0.53 0.43 0.45 0.55
a-b Means in a given row bearing different superscripts differ significantly at P < 0.05.
1 Values given in this table correspond to least-squares means obtained from ANOVA and their pooled SEM.
2 n = 10 birds per mean
5
64.9 b
70.2 b

Shrimp Industry Organic Wastes
14 d - old broiler chicks
6

Literature Cited
Association of Official Analytical Chemists. AOAC. 1990. Official Methods of Analysis. Williams, J.
(ed). Arlington, VA.
Cunha, F. S., C. B. V. Rabello, M. C. M. M. Ludke, W. M. Dutra, R. R. S. Loureiro, R. C. Lucas, L.
Piaggio, and E. R. Prates. 2003a. Use of shrimp meal in the feeding of broiler chickens. IX
World Conference on Animal Production. Porto Alegre, Brasil.
Cunha, F. S., C. B. V. Rabello, M. C. M. M. Ludke, W. M. Dutra, V. R. B. A. Rocha, C. R. G. de
Freitas, and F. B. Lima. 2003b. Effect of shrimp meal on carcass yield in broiler chickens. IX
World Conference on Animal Production. Porto Alegre, Brasil.
Damron, B.L., P.W. Waldrup, and R.H. Harms. 1964. Evaluation of shrimp meal in broiler diets.
Poultry Science Mimeograph Series No. PY65-1. University of Florida, Gainesville, Fl.
Ewing, R., 1963. Handbook of Poultry Nutrition. J.J. Little and Ives Co. South Pasadena, CA.
Gernat, A.G. 2001. The effect of using different levels of shrimp meal in laying hen diets.
Poultry Sci. 80: 633-636.
Ilian, M.A., C.A. Bond, A.J. Salam, and S. Al-Hooti. 1985. Evaluation of shrimp by-catch meal as
broiler feedstuff. Nutr. Rep. Int. 31: 487-492.
Islam, M.A., M.D. Hossian, S. M. Baibul, and M.A.R. Howlider. 1994 Unconventional feed for
broilers. Indian Vet. J. 74: 775-780.
Meyers, S.P. and J.E. Rutledge, 1971. Shrimp meal – A new look at an old product. Feedstuffs
43 (49):31.
National Research Council (NRC), 1994. Nutrient Requirements of Poultry. 9 th rev. ed.
National Academy Press, Washington, D.C.
Rosenfeld, D.J., A.G. Gernat, J.D. Marcano, J.G. Murillo, G.H. Lopez, and J.A. Flores. 1997. The
effect of using different levels of shrimp meal in broiler diets. Poultry Sci. 76: 581-587.
SAS Institute, 1990. SAS ® / STAT guide for personal computers. Version 6.12 edition. SAS
Institute Inc. Cary, NC.
Singletary, J. R., D.J. Bray, and H.J. Davis. 1935. Shrimp meal as a protein supplement for
chickens. Page 31 in: Bulletin 262. Louisiana State University, Baton Rouge, LA.
7

Viability of poultry litter compost on growth of Brachiaria brizantha cv. Mulato and pollutant
effects in the soil
Emérita García Pizarro and Elide Valencia
Department of Agronomy and Soils, University of Puerto Rico, Mayagüez
Abstract
Composted poultry litter (CPL) applied to field crops can be an important source of nutrients and organic
matter to improve soil quality. However, little is known on the effects of CPL on tiller dynamics and
forage yield of the tropical grass Brachiaria brizantha cv. mulatto. A study was conducted in the fall of
2003 and spring of 2004 to evaluate four CPL rates and their influence on number of tillers, herbage
mass and crude protein (CP) concentration of cv. Mulato at three successive harvests at 30-d intervals.
Treatments included 0 (Control; C), 16 (low; L), 31 (Medium, M), and 62 kg/ha of N (High; H) CPL rates.
Grass seedlings (6-wk old) were established in pots (1-gl) filled with soil of the Consumo type (Fine,
mixed, semi-active, isohyperthermic Typic Haplohumults). Composted poultry litter was broadcastapplied
and incorporated into the first 2-cm of the soil in the pots. Plants were irrigated as needed. From
first to third harvest, there was a minimal increase in tiller count (8.4 to 11) for the control.
Corresponding increases observed for the L and M CPL rates were 14 to 28 and 21 to 28 tillers/plant.
However, the magnitude of change in tiller counts at the H CPL rate was much greater (21, 23, and 44)
at first, second and third harvest, respectively. There was also a linear effect (P

Introduction
The local livestock industries are dependent on tropical pastures to meet their nutritional
demand. Nitrogen is the most limiting nutrient for pasture production in Puerto Rico. Most of
the soils used for pastures are mainly acidic (Oxisols and Ultisols). These soils have low organic
matter content, and are prone to erosion. Grass production relies on inorganic fertilizers, thus
increasing cost of production. Nitrate leaching and subsurface lateral flow toward streams and
rivers is a major cause of contamination of groundwater and other water resources. Phosphorus
is also a major concern (Havlin et al., 1990). According to Hornik, (1992), the quality of soil
has traditionally been associated mainly with its productivity. This definition has now been
expanded to include the capacity of a soil to function within ecosystem boundaries to sustain
biological productivity, maintain environmental quality, and promote plant and animal health.
An alternative for improving soil quality is the use of compost.
Poultry litter (PL) is generated in large quantities on the island. The Department of Agriculture
of Puerto Rico reports an average yearly population of 8.2 million chickens which yield
approximately 71,500 metric tons of manure (Muñoz et al., 1990) which represent an
alternative to use for organic matter maintenance of low fertility soils. According to Martinez
and Guzman (1999), composting is a cost effective and environmentally sound alternative for PL
recycling. On clay soils, good quality compost can improve soil structure, reduce surface
crusting and compaction, promote drainage, and provide much needed nutrients. In sandy soils,
compost increases water and nutrient retention, supplies nutrients, and increases microbial
activity.
In an evaluation of time and mode of application of chicken manure for plantain production, an
adverse effect was observed during germination with the application of 7.26 kg manure per
plant at planting (Muñoz and Martínez, 1991). In other studies PL was shown to promote faster
turf establishment, improve turf density and color, increase root growth, and lower the
requirements of inorganic fertilizer and irrigation (Landschoot, 1996). Composts can supply all
or most of the turf’s nutrient requirement (i.e. N, P, S, K, Fe, Zn, Cu, and Mg). Studies with
biosolids show that only about 10 percent of the N is available to plants during the first growing
season. Certain types of compost have a high concentration of soluble salts, such as those
made with spent animal manures. Those salts increase the electrical conductivity of the soil with
compost additions, sometimes approaching limits considered detrimental to crop growth
(Martínez and Guzmán, 1999; Landschoot, 1996). Another problem is its variable pH as very
9

high (>8.5) or very low (

Results and Discussion
Table 1. Composition of soil analyzed at two depths (Fine, mixed, semiactive, isohyperthermic
Typic Haplohumults) and of composted poultry litter (CPL).
Sample Organic Matter * K * Mg * Ca * N * P ** CEC *** pH
Soil
0-4” depth 2.1 3.1 21.1 49.6 .2 3 21.4 5.5
4-6” depth 2.6 6.7 25.2 57.7 .2 21 22.2 6.3
Poultry Litter 9.9 65.1 14.8 20.1 2 238 46.3 9.1
* Values are in percent
** P values are in ppm
*** Values are in meq/100g
Table 2. Micro-nutrients and sulfur composition of soil at two depths and of composted poultry
litter (CPL).
Metal (ppm) 0-4Ӡ 4-6Ӡ
Soil CPL
Sulfur 139 322 964
Zinc 2.4 5.4 58.9
Manganese 55 84 126
Iron 31 39 60
Copper 3.9 4.5 4.9
Boron 0.3 0.8 3.8
† Soil depth
There were significant (P

was minimal increase in number of tillers (8.4 to 11) in the control treatment without CPL.
Similar increases were observed for the L and M rates. Major changes, however, were noted
for the H rate (21, 23, and 44 tillers per plant) at first, second and third harvest, respectively
(Figure 1).
Figure 1. Number of tillers per plant for four CPL treatments at 30 (A), 60 (B) and 90-d (C)
Treatments
High
Medium
Low
Control
harvests.
0 20 40 60 80 100
Yield gram/plot
A B C
Figure. 2. Dry matter yield (g/plot) for four CPL treatments at 30 (A), 60 (B) and 90-d (C)
harvest..
12

There was also a linear effect of CPL rates on dry matter (DM) yield (g/plant). For the control,
there were no changes in DM yield from first to last harvest (average of 16 g per plant). For
the L and M rates of CPL minimal changes in dry weight of plants were observed from the first
to third harvest. However, at the H rate of CPL, there were major changes in dry matter yield.
Yields per plant of 31, 56, 81 g at first, second, and third harvest, respectively were recorded
(Figure 2). The increase in DM at third harvest is highly correlated to number of tillers (44
tillers/plant). There were differences in CP concentration of cv. Mulato with increasing
application of CPL. Crude protein averaged 7.2, 6.6, 7.2, and 9.1% for the C, L, M, and H rates,
respectively. Thus there was nearly a 2 percentage units increase in CP with the H rate of CPL
over C.
Conclusions
Our results show positive linear responses for both number of tiller and DM yield of cv. Mulato
to the high rate of CPL (62 kg/ha N). The increased DM yield due to the H CPL rate compared
to that of the control at the third harvest may indicate mineralization of N and more N available
for plant use. This gives CPL the quality of a slow release fertilizer. The two percentage units
increase observed in CP with addition of the H rate of CPL indicates that CPL could serve to
increase the nutritive value of cv. Mulato. Soil analysis is needed to determine the
corresponding changes in soil pH, and P and other mineral accumulations in the soil. Because
of the slow release of N from CPL, there would be less leachate of N in the system. An
important aspect of this study is that cv. Mulato or other tropical grasses can serve as a sink for
this nutrient-rich compost and also provide an environmentally acceptable and hopefully use of
poultry litter compost.
13

Brachiaria brizantha cv. Mulato Mulato grass seedling (6 -wks old).
Brachiaria brizantha first day of experiment Brachiaria brizantha on the 30 th day.
Plant appearance after each treatments, Control, Medium, Low and High rates
14

Literature Cited
AOAC. 1990. Official Methods of Analysis. 15 th Ed. Association of Official Analytical Chemist.
Arlington, VA.
Havlin JL, D.E. kissel DE, and L.D Maddux. 1990. Crop rotation and tillage effects on soil organic
carbon and nitrogen. Soil Sci. Soc.Am. J. 54: 448-452.
Hornik SB (1992) Factor affecting the nutritional quality of crops. Am. J. Altern. Agric 7:63-68
Landschoot, P. 1996. Using compost to improve turf performance. Pennsylvania State
University. Technical Bulletin. College of Agricultural Sciences Cooperative Extension Services.
Martinez, G. and J.L. Guzman. 1999. Chemical and physical properties of two tropical soils
treated with sewage sludge compost. J. Agric. Univ. PR. 83:103-121.
Muñoz, M. A., and G. Martinez, 1991. Chicken, manure: An organic fertilizer for plantains ( Musa
accuminata x M. balbisiana AAB). Proc. Caribbean Food Crops Society 27:225-233, Roseau,
Dominica.
Muñoz, M., O. Colberg and J.A. Dumas. 1990. Chicken Manure as an Organic
Fertilizer.J. Agric. Univ. P.R. 74: 139-145.
SAS Institute. 1990. SAS/STAT® User’s Guide (Release 8.1). SAS Inst. Inc., Cary, N.C. 1686
pp.
15

Fermentation characteristics and aerobic stability of orange pulp silage treated with urea
Jonael Bosques Méndez, Abner A. Rodríguez and Danilo Cianzio
Department of Animal Industry, University of Puerto Rico, Mayagüez Campus
Abstract
The objective of this study was to determine the fermentation characteristics of orange pulp (OP) treated
with urea. The aerobic stability of fresh and fermented OP was also evaluated. The four treatments
tested were OP without urea and with 1, 3, and 5% urea (w/w). Mixtures were fermented (N=3) in
micro-silos for 30 and 60 d. Samples from each treatment were collected at 0, 30 and 60 d to determine
pH, chemical composition and fermentation products. Results showed that it is possible to ferment OP
for 30 or 60 d without affecting the original chemical composition of the fresh residue. After 30 and 60 d
of ensiling, acetic acid content was similar (P

Introduction
Orange pulp (OP) is an organic waste resulting from the operation of a juice processing plant.
In Puerto Rico, this by-product has been partially used in direct feeding to dairy cows, or
otherwise disposed of in landfills, a practice that represents increased operational costs for the
industry and a source of environmental pollution for the island. The orange juice plant
“Productores de Citrícos de la Montaña”, located in the center of the island, produces in a
normal day of operation 25m 3 of wet citrus pulp. Therefore, alternatives for the disposal of this
by-product need to be evaluated. Silage production has been identified as one such possible
alternative. However, the combined effect of high water soluble carbohydrates content of the
citrus pulp and the high temperatures of the local tropical environment could lead to rapid
deterioration of the fresh material, an undesirable type of fermentation (alcoholic), and poor
aerobic stability of the fermented waste. The practice of adding a non-protein nitrogen source
has been reported to improve the fermentation characteristics and aerobic stability of grass
silage (Woolford, 1990), but there is limited information regarding the use of urea as an
additive on the ensiling characteristics of OP. The objective of this study was to determine the
fermentation characteristics of OP silage treated with 3 levels of urea. The aerobic stability of
fresh and fermented OP was also determined.
Materials and Methods
The study was conducted in the Animal Nutrition Laboratory, University of Puerto Rico,
Mayagüez Campus. The organic waste was obtained from the “Productores de Cítricos de la
Montaña” processing plant. For the fermentation studies, OP was fermented alone or mixed
with urea (non-protein nitrogen source) at three different levels; 1, 3, and 5 % (w/w). Mixtures
were allowed to ferment in PVC lab micro-silo (1.2 kg capacity) for 30 and 60 days (Fig. 1).
Triplicate samples from each treatment and length of fermentation were analyzed to determine
pH, chemical composition (AOAC, 1991), and fermentation end-products. Data were analyzed
as a completely random design with a 4 (levels of urea, 0, 1, 3, and 5 %) by 3 (length of
fermentation, 0, 30, and 60 d) factorial arrangement of treatments (Steel and Torrie, 1980)
using the General Lineal Model subroutine of SAS (1990). The Bonferroni t-test was used for
separation of means. To determine the aerobic stability of the fresh residue and fermented
product treated with or without urea, triplicate samples (2 kg) of fresh or fermented OP were
place into styrofoam containers lined with plastic and exposed to air for 6 days (Fig. 2).
Samples were collected at 0, 1, 3, 6 (fermented) and 9 (fresh material only) d to determine pH,
17

and yeast and mold (YM) populations. Temperature was monitored daily. Fresh and fermented
material recovery percentage was calculated form each time of exposure. Statistical analysis of
the pH and YM populations data was performed as a completely randomized design with a 4
(levels of urea) by 4 or 5 (periods of aerobic exposure) factorial arrangement of treatments
(Steel and Torrie, 1980) using the General Lineal Model subroutine of SAS (1990). The models
for temperature and OP recovery percentage were similar except that 7 (temperature) and 3 (
fermented) or 4 (fresh) periods of aerobic exposure were utilized, respectively. For all models,
mean separation was performed by Bonferroni t-test.
Fermentation Characteristics Results and Discussion
For both lengths of fermentation pH was lower (P

During the whole aerobic exposure period pH was lower (P

Fresh Orange Pulp
Anaerobic Fermentation (30 and 60 days)
Orange Pulp Silage
Figure 1. Orange pulp ensiling process
20

Table 2. Stability of fresh and fermented orange pulp treated with urea exposed to aerobic
conditions during 6 days.
Component Aerobic exposure (d) Fresh Orange Pulp
pH 0 3.76
1 3.74
3 3.71
6 3.38
9 3.23
Temperature, °C 0 25.53 d
1 38.88 a
3 37.40 b
6 30.37 c
9 32.59 c
Yeast and Molds (ufc/g) 0 4.39 c
1 6.15 b
3 7.71 a
6 6.52 b
9 7.68 a
Organic Pulp Recovery (%) 1 95.83 a
3 87.30 b
6 82.00 bc
9 77.93 c
Urea (%)
Control 1 3 5
pH 0 3.36 c 3.78 b 4.05 ab 4.15 a
1 3.33 c 3.75 c 4.00 c 3.92 c
3 4.00 b 4.34 a 4.30 a 4.38 a
6 3.44 c 4.63 b 4.67 b 5.28 a
Temperature, °C 0 22.00 22.16 22.01 22.37
1 27.04 27.22 26.50 26.50
3 32.22 a 32.38 a 30.42 b 27.43 b
6 29.66 a 31.67 a,b 30.19 a 28.75 b,c
Yeast and Molds (ufc/g) 0 3.05 a 3.83 a 2.57 b 1.45 c
1 5.67 a 3.99 b 2.17 c 1.82 d
3 8.25 a 5.53 c 6.63 b 5.78 c
6 7.16 c 8.83 b 9.58 a 9.16 a
Organic Pulp Recovery (%) 1 88.56 90.00 86.43 89.86
3 87.76 88.09 88.42 89.42
6 85.21 84.44 87.04 86.25
a,b,c, Means in the same row differ (P

Day 0
Day 3
Day 9
Day 1
Day 6
Figure 2. Deterioration of fresh orange pulp exposed to air during 9 days
22

Conclusions
It is possible to ferment OP during 30 or 60 d without greatly affecting the nutritive profile of
the fresh product. Deterioration of fresh OP increased directly with length of aerobic exposure.
Addition of urea improved the quality of the silage and its aerobic.
Literature Cited
AOAC. 1991. Official Methods of Analysis. Association of Official Analytical Chemist, Washington,
D.C.
Crawshaw, R. 2001. Co-product Feeds. Animal feeds from the food and drinks industries.
Nottingham University Press. Nottingham, NG11 OAX, UK.
SAS (1990). SAS User’s Guide: Statistics. SAS Inst., Cary, N.C.
Ting, S.V. and J.A. Attaway. 1971. Citurs fruits. In: The biochemistry of Fruits and their
Products. Vol-2, ED. A.C. Hulme, Academic Press.
Woolford, M.K. 1990. The detrimental effect of air on silage. J. Appl. Bact. 68:101.
23

Fermentation and aerobic stability of silages made of rice hulls hydrated with milk
Maritere Crespo, Angel A. Custodio and Abner A. Rodríguez
Department of Animal Industry, University of Puerto Rico, Mayagüez
Abstract
In Puerto Rico, milk (M) that does not meet minimum standards for human consumption is rejected and
dumped in the waste lagoons of dairy farms, which contributes to pollution and constitutes a loss of
unutilized nutrients. Rice hulls (RH), although of low nutritional value, are in abundant supply and
accessible especially in the most intensive dairy farming zone of the island and may be used to recycle
the milk as silage. The purpose of the present study was to evaluate the quality and aerobic stability of
silages made from rice hulls hydrated with different levels of milk. The experiment was performed with
seven treatments: 1) Control (39%water 61% rice hulls w/w), 2) 35M:65RH, 3) 42M:58RH, 4)
45M:55RH, 5) 50M:50RH 6) 60M:40RH and 7) 70M:30RH w/w, ensiled in micro-silos of PVC pipes in
triplicate. Silos were opened after 14 and 28 days. To evaluate quality, pH and fermentation products
were measured in three samples from each treatment, at 0, 14 and 28d. For the aerobic stability
determination, pH and temperature were measured the day the silo was opened and for the next three
days. An analysis of variance was performed with treatment and days of fermentation as independent
variables. Results showed a pH level of about 3.8 at 14d and 3.9 at 28d for all treatments with 50%M
or more. Major fermentation product was lactic acid in all treatments except the control, in which
butyric acid was more abundant. Lactic acid concentration was highest (P

los 14 días. Las concentraciones de los ácidos butírico, propiónico, acético e isobutírico permanecieron
por debajo de 0.5% en todos los tratamientos. A las 24 horas de exposición al aire la temperatura
aumentó de 6 a 17°C (P

ambient temperature that ranged from 27 to 30 °C. Triplicates were prepared for each
treatment and sampled at 0, 14 and 28 days of fermentation. Samples were analyzed for pH
immediately after opening each silo (Figure 3) and frozen to be sent later to Dairy One Forage
Lab in Ithaca, New York, for determination of fermentation end-products.
To determine the stability of fermented rice hulls to aerobic exposure, 500g samples of silage
were placed in styrofoam containers lined with plastic bags (Figure 4) and exposed to air for 3
days. Temperature of the silage was measured at days 0, 1, 2, and 3 while pH was measured
at days 0, 1, and 3 of aerobic exposure. Dry matter recovery at days 1 and 3 of aerobic
exposition was calculated as the difference in total dry matter relative to the initial quantity.
Analyses of variance with treatment and days of fermentation as independent variables were
performed using SAS GLM procedure (SAS, 1990). Differences among means were evaluated
using Bonferroni comparisons.
Fermentation Characteristics Results and Discussion
The high pH and very low acetic acid content (Table 1) of the control silage demonstrate that it
did not ferment well. Relatively high butyric acid of this silage is indicative of protein
degradation due to lack of available carbohydrates in rice hulls. For all treatments with 50% of
milk or more, average pH level was about 3.8 at 14d and 3.9 at 28d. Major fermentation
product was lactic acid in all silages except the control, in which butyric acid although in low
concentration, was the most abundant. Lactic acid concentration was highest (P

emained high at days two and three. The increment of temperature upon aerobic exposure
was directly related to the level of milk added, with the highest increase observed in silage
with 70%M. After three days of aerobic exposure silages that contained milk were badly
damaged. Thus, mixtures of rice hulls and milk in proportions ranging from 65:35 to 40:60
showed a good fermentation, but the resulting silages deteriorated in less than 3 days upon
aerobic exposure.
Table 1. Chemical analysis at 14 and 28 days of fermentation of silages from rice hulls hydrated
with varying proportions of milk
Organic 2
Inorganic 2 Lactic 2 Acetic 2 Butyric 2
Treatments 1 DM (%) pH matter matter acid acid acid
Fermentation Length (14 d)
Control 56.64 5.17 84.30 15.64 0.02 0.00 0.20
2 59.49 3.84 85.05 14.94 1.26 0.14 0.00
3 59.55 4.02 85.56 14.43 1.32 0.14 0.02
4 56.07 3.97 85.16 14.83 1.38 0.14 0.02
5 54.91 3.80 85.90 14.10 2.16 0.15 0.02
6 48.67 3.78 84.91 15.08 1.91 0.10 0.02
7 34.91 3.68 85.94 14.06 1.76 0.09 0.01
Fermentation Length (28 d)
Control 51.13 4.97 83.66 16.33 0.00 0.16 0.32
2 61.89 4.06 84.81 15.19 1.34 0.09 0.02
3 59.57 4.04 85.97 14.69 1.47 0.13 0.08
4 56.62 4.02 85.09 14.91 1.47 0.15 0.02
5 56.85 3.75 85.27 14.72 2.11 0.18 0.04
6 51.03 3.93 84.14 15.85 1.99 0.10 0.03
7 46.23 3.74 84.71 15.28 1.56 0.09 0.01
1 Control had 39% water and 61% rice hulls, treatments 2,3,4,5,6, and 7 had 35,42,45,50,60, and 70% milk (w/w), respectively.
2 Dry matter basis
Table 2. Change in pH over three days of aerobic exposure of silages made of rice hulls
hydrated with varying proportions of milk
Days of air Treatments
exposure Control 2 3 4 5 6 7
Fermentation Length (14 d)
0 5.17 3.84 4.02 3.97 3.80 3.78 3.68
1 5.19 4.77 4.10 4.05 3.92 5.75 5.95
3 6.69 6.34 6.52 6.61 6.26 8.05 7.69
Fermentation Length (28 d)
0 4.97 4.06 4.04 4.02 3.75 3.93 3.74
1 4.78 4.12 4.08 3.97 3.83 3.81 5.53
3 6.76 6.21 5.48 6.42 4.66 6.68 7.84
1 Control had 39% water and 61% rice hulls, treatments 2,3,4,5,6, and 7 had 35,42,45,50,60, and 70% milk (w/w), respectively
27

Table 3 Temperature (°C) change from day 0 to day 3 of aerobic exposure silages made of rice
hulls hydrated with varying proportions of milk
Days of air Treatments
exposure Control 2 3 4 5 6 7
Fermentation Length (14 d)
0 25.40 25.76 25.93 25.26 25.55 25.92 25.55
1 26.66 37.40 32.23 30.76 37.40 41.10 39.99
2 30.00 28.51 34.06 34.03 28.51 38.88 44.07
3 30.36 29.25 30.76 33.13 29.25 37.58 36.66
Fermentation Length (28 d)
0 24.03 25.40 25.60 25.36 24.74 24.25 24.81
1 26.50 32.20 32.23 26.33 28.15 32.40 45.18
2 33.30 34.23 33.53 33.86 28.88 32.40 44.62
3 29.43 31.10 35.16 32.20 37.03 43.51 42.03
1 Control had 39% water and 61% rice hulls, treatments 2,3,4,5,6, and 7 had 35,42,45,50,60, and 70% milk (w/w), respectively
Fig. 1. Rice Hulls Fig. 2. Micro silos made of PVC pipes
Fig. 3. Opening silos after 14 and 28 days of Fig. 4. Sample of silage (500 g) placed
Fermentation in Styrofoam containers lined with plastic
bags for the aerobic exposure period
28

Conclusions
The mixture of milk to rice hulls in nearly equal proportions is conducive to a good
fermentation, but the resulting silage deteriorates rapidly upon aerobic exposure. Ensiling milk
with other more nutritive materials, such as straw or hay, should be even more successful as a
method of preserving the nutrients of discarded milk for animal feeding and thus contributing
to reducing soil and water contamination
Literature Cited
Wienberg Z.G., G. Ashbell, and Y. Chen. 2003. Stabilization or returned dairy products by
ensiling with straw and molasses for animal feeding. J. Dairy Sci. 86:1325-1329.
González G. and A. A. Rodríguez. 2003. Effect of storage method on fermentation characteristic,
aerobic stability, and forage intake of tropical grasses ensiled in round bales. J. Dairy Sci. 86:
926-933.
Rodríguez A. A., J. A. Acevedo, and E. O. Riquelme. 1997. Aerobic Stability of Native Tropical
Grass Silage. Effect of propionic acid and length of aerobic exposure. Arch. Latinoam. Prod.
Anim. 5(Supl. 1): 83-85.
SAS Institute. 1990. SAS User’s Guide: Statistics. SAS, Inst., Cary, N.C.
Oficina de la Reglamentación de la Industria Lechera. 2002. Informe Anual Año Fiscal 2001-
2002. ORIL, Hato Rey, P.R.
29

Development of Fermented Liquid Diet Using Organic Wastes For Early Weaned Pigs
Dianne Hernández, Paul F. Randel, and Abner A. Rodríguez-Carías
Department of Animal Industry, University of Puerto Rico, Mayagüez Campus
Abstract
An experiment was conducted to determine the fermentation characteristics of a commercial weaning pig
concentrate (CPC) mixed with liquid organic wastes from the pharmaceutical (SynerMax, SM), beverages
(wastewater from a caramel processing plant; WWCP) and food industries (milk whey, MW and discarded
milk, DM). The CPC physical form (pelleted, CPCP or grounded, CPCG) and the addition of a lactic acidproducing
bacterial inoculant (LAPBI) were also evaluated. The experiment consisted of mixing the CPC
(P or G) with the liquid wastes in a 2:1 (w/w) proportion and with (LAPBI) or without the bacterial
inoculum (NLAPBI). Deionized water (DW) was used as control. Mixtures evaluated were SM-CPCP-
NLAPBI, WWCP-CPCP-NLAPBI, MW-CPCP-NLABPI, DW-CPCP-NLAPBI, DW-CPCG-NLAPBI, DW-CPCG-
LABPI, DM-CPCG-NLAPBI, and DM-CPCG-LABPI. To determine the fermentation characteristics triplicate
samples from each mixture were placed in hermetic anaerobic digestors (1000 mL capacity) during 96
hours. Samples (50 g) from each mixture were taken at 0, 12, 24, 48, 72, and 96 h to determine pH and
fermentation products (organic acids). During the entire fermentation period and for all mixtures,
production of propionic, and butyric acid was minimal, reaching concentrations lower than 0.02%. After
96 h, mixtures containing SM, WWCP, and MW did not ferment satisfactorily. Fermenting DW with CPCG
resulted in lower (P

Introduction
Disposal of liquid by-products generated during manufacturing processes in the pharmaceutical
and food industries represents an increase in operating costs and environmental pollution for
the island. SynerMax or Streptomyces soluble concentrate is an enzyme-treated, nutrient rich,
liquid, co-product from Abbott Laboratories’ fermentation manufacturing which uses stringently
pre-tested edible–grade soy-based ingredients. When blended into livestock diets, it provides a
unique source of digestible proteins, lipids, carbohydrates, minerals, vitamins and unidentified
response factors. However, more research is needed to prove its potential use in livestock diets.
Waste water resulting from the caramel production is another industrial by-product that has
been added in growing pigs’ diets as an energy source without affecting animal performance
(León, 2002; Jiménez, 2003). However, and similar to Synermax, more experimental data are
needed for its optimal inclusion in animal diets. Milk whey is a liquid by-product of cheese
production consisting mostly of water, lactose, minerals and a small amount of the more soluble
protein fractions that has been used in animal diets with variable success; but similar to
discarded milk (milk that does not meet minimum standards form human consumption) it is
presently dumped in wastes lagoons. Recycling of nutrients form these liquid organic wastes
into animal diets represents an alternative to reduce costs of production and environmental
pollution. In the feeding management of postweaning pigs, the use of fermented liquid diets
utilizing certain agro-industrial by-products has been evaluated with great success, increasing
animal performance and health (Scholter, 2001). However, there is little information regarding
the use of local liquid agro-industrial by-products to develop a liquid diet for pigs.
Objective
To determine the fermentation characteristics of a commercial weaning pig concentrate mixed
with liquid organic wastes resulting from the pharmaceutical, beverages, and food producing
industries.
Materials and Methods
The study was conducted in the Animal Nutrition Laboratory, University of Puerto Rico,
Mayagüez Campus. A commercial pig concentrate (CPC) was mixed with each of four liquid
industrial organic wastes. By-products evaluated were SynerMax (SM), waste water from a
caramel plant (WW), milk whey (MW) and discarded milk (DM). A mixture of deionized water
(DW) CPC was used as a control treatment. The physical form of the CPC (pelleted, CPCP or
31

ground, CPCG; 1 mm screen) and the use of a lactic acid-producing bacterial inoculant (LAPBI)
as microbial starter applied at 10 6 cfu/g or not (NLAPRB) were also evaluated. Mixtures
evaluated include 1) SM-CPCP-NLAPBI, 2) WWCP-CPCP-NLAPBI, 3) MW-CPCP-NLAPBI, 4) DW-
CPCP-NLAPBI, 5) DW-CPCG-NLAPBI, 6) DW-CPCG-LAPBI, 7) DM-CPCG-NLAPBI, and 8) DM-
CPCG-LAPBI. The CPC was mixed with the liquid wastes in 2:1 proportions (w/w). To
determine the fermentability of the different mixtures, triplicate samples from each treatment
were placed into six hermetic digesters (1,000 ml capacity), shown in Fig. 1, and kept at
ambient temperature (27-30°C). Samples (50 g) were taken from each digester at 0, 12, 24,
48, 72 and 96 hours and analyzed for pH and organic acids. For pH determination, 50 g of the
mixture were placed into 450 ml of distilled water (w/v) and homogenized for 5 min with a
stomacher apparatus. Homogenates were strained through eight layers of cheesecloth and
analyzed with a pH meter fitted with a combination electrode that was standardized from pH 4
to 7 using commercial buffers. Fermentation products (lactic, acetic, propionic, and butyric
acids) were quantified in a commercial certified laboratory. Data were analyzed as a completely
randomized design with a 8 (mixtures) by 6 (fermentation periods 0, 12, 24, 48, 72, and 96 h)
factorial arrangement of treatments using the General Lineal Model subroutine of SAS (1990).
The Bonferroni t-test was be use for separation of means.
Results and Discussion
Information regarding the initial chemical composition of the liquid organic wastes tested in this
experiment obtained from various sources is presented in Table 1. An acid pH and a lactic acid
content greater than 2%, is characteristics of the by-product from the pharmaceutical industry
(SynerMax). The waste water from the caramel processing plant is less acidic than SM, but also
has a greater lactic acid content (1.4 %) than typical liquid organic wastes from the food
industry.
During the fermentation for all mixtures production of propionic, and butyric acids was minimal,
reaching concentrations lower than 0.02%. Likewise, during 96 hours of fermentation no
changes (P

liquid density, high concentration of minerals (e.g. S, Na), and organic acids affect the activity
of desirable bacteria associated with the fermentation process.
Table 1. Chemical composition of liquid organic wastes evaluated
Item (%) SynerMax Wastewater from a caramel plant Milk Whey 1 Discarted Milk 1
Dry Matter 48.7 12.7 4.01 13.20
Organic Matter 2 91.6 75.6 99.0 99.0
Inorganic Matter 2 8.4 24.4 .05 1.0
Crude Protein 2 14.5 5.90 1.02 3.80
Crude Fat 2 8.4 2.70 1.45 2.90
Lactose ND ND 2.45 4.50
pH 3.81 5.27 6.52 6.40
Lactic acid 2.04 1.41 ND ND
Acetic acid 1.00 0.00 ND ND
Propionic acid 0.27 0.00 ND ND
Butyric acid 0.00 0.00 ND ND
1 As fed, ND = Not determined
In CPC pelleted and fermented with milk whey (mixture 3, table 2) the pH decreased (P< .05)
and lactic acid and acetic acid contents increased (P < .05) as length of fermentation increased.
However, final pH was higher and lactate concentration of the mixture was lower than the
expected values indicative of well fermented feedstuffs. Grinding of the CPC seemed to
influence positively the fermentation process. Grounded CPC mixed with deionized water
(mixture 5) had lower (P

Table 2. Characteristics of liquid organic wastes fermented with a commercial pig concentrate during 96 hours
Fermentation Product (g/100g)
Mixture Components Physical Form 1 LAPBI 2 Period (h) pH Lactic Acetic
1 SM:CC P No 0 5.38 1.36 0.21
12 5.67 1.30 0.19
24 5.73 1.51 0.23
48 5.77 1.44 0.32
72 5.71 1.39 0.29
96 5.77 1.50 0.33
2 WW:CC P No 0 4.46 0.43 0.06
12 4.50 0.59 0.09
24 4.50 0.56 0.08
48 4.45 0.44 0.06
72 4.40 0.73 0.11
96 4.42 0.52 0.11
3 MW:CC P No 0 6.16 a 0.05 c 0.01 b
12 6.17 a 0.10 c 0.01 b
24 6.14 a 0.13 bc 0.02 b
48 5.94 b 0.10 c 0.09 a
72 5.73 c 0.17 b 0.12 a
96 5.30 d 0.27 a 0.09 a
4 DW:CC P No 0 6.35 a 0.07 c 0.00
12 6.13 b 0.18 c 0.00
24 6.14 b 0.07c 0.01
48 5.77 c 0.19 c 0.01
72 5.33 c 0.32 b 0.09
96 4.77 d 0.70 a 0.12
5 DW:CC G No 0 6.19 a 0.08 c 0.05 c
12 6.25 a 0.15 c 0.06 c
24 6.15 ab 0.17 c 0.19 a
48 6.10 b 0.21 bc 0.26 a
72 5.69 c 0.35 bc 0.11 b
96 4.61 d 0.92 a 0.14 b
6 DW:CC G Yes 0 6.39 a 0.08 d 0.01
12 6.31 a 0.29 c 0.01
24 6.34 a 0.38 c 0.01
48 5.90 b 0.60 b 0.02
72 5.49 d 1.02 a 0.06
96 4.57 e 1.08 a 0.09
7 DM:CC G No 0 6.44 a 0.09 d 0.01 b
12 6.30 b 0.11 d 0.01 b
24 6.24 b 0.66 c 0.02 b
48 5.61 c 0.72 c 0.16 a
72 4.70 d 1.22 b 0.20 a
96 4.44 e 2.89 a 0.23 a
8 DM:CC G Yes 0 6.45 a 0.09 d 0.01 b
12 6.28 b 0.10 d 0.01 b
24 6.24 b 0.09 d 0.01 b
48 5.47 c 0.73 c 0.03 b
72 4.79 d 2.10 b 0.18 a
96 4.42 e 3.03 a 0.23 a
Expected Final Value < 4.5 < 1.5 >.80
1 P = Pelleted; G = Grounded
2 LABPI = Lactic acid-producing bacterial inoculant
a,b,c,d,e Means with unlike within mixture superscripts differ (P < .05)
34

Fig. 1. Anaerobic digestors fermenting liquid organic wastes with a commercial early pig concentrate
Literatured Cited
Jiménez, E. 2003. Use of Waste Waters from a Caramel Producing Plant as a Supplement in the
Diet of Early Weaning and Finishing Pigs. M.S., Department of Animal Industry, University of
Puerto Rico, Mayagüez Campus. 57 pp.
León, F.J., R. Sánchez, C.S. Santana and A. Rodríguez. 2003. Waste Water from a Caramel
Industry as a Partial Substitute in Diets for Pigs. Journal of Agriculture of the Unversity of
Puerto Rico. 87 (1-2): 87-90.
SAS/STAT. 1990. SAS User’s Guide (Release 6.12). SAS Inst., Inc., Cary, NC.
Scholten, R.H.J. 2001. Fermentation of liquid diets for pigs. Ph.D. Dissertation, Wageningen
Institute of Animal Science, Department of Animal Nutrition. Wageningen University, The
Netherlands.
35

Fermentation characteristics and dry matter disappearance of grass hay, rhizome peanut
and by products silages
Karla M. Tous, Teodoro Ruiz and Héctor L. Díaz
University of Puerto Rico, Mayagüez Campus, Mayagüez, Puerto Rico 00680
Abstract
In situ dry matter disappearance (ISDMD) was determined on four feeds in both the fermented and
unfermented state: residues of tilapia (Oreochromis niloticus) fillet production, pineapple processing
residues (Ananas camosus), rhizome perennial peanut hay (Arachis glabra a) t and stargrass (Cynodon
nlemfuensis) hay, for which two fistulated Holstein cows fed grass hay were used. Triplicate samples
from each feed, in Dacron bags were intraruminally incubated for 48 hrs, and then analyzed for DM
disappearance. Data were analyzed using the general linear model of SAS® and Bonferroni test for
separation of means. Results showed significant differences in dry matter content of the fermented
feeds, rhizome peanut being highest (35.61%) and pineapple residue lowest (10.35%), (P

Introduction
One of the most important and limiting aspects to consider in ruminant livestock production is
the type and quality of feed provided. Use of high quality feeds can translate into higher
productions of milk and meat. This is especially important in the tropics, were ruminants
experience environmental conditions that limit their abilities for feed consumption and growth.
The dependence on low quality tropical forages as ruminant feeds makes it difficult to achieve
the genetic potential of high producing animals. Silages from legume forages like rhizome
perennial peanut, constitute a viable alternative to provide animals the high quality feeds they
require. These forages are characterized by lower concentrations of fiber, and higher
concentrations of degradable nutrients, particularly in crude protein (CP), relative to grass hay.
Grass hay tends to present higher concentrations of fiber and lower rates of digestibility.
Perennial peanut is a high quality, persistent legume that adapts easily to tropical and sub-
tropical environments (Staples et al., 1997). Silages made from organic residues, such as those
resulting from processing of tilapia, are quite high in protein content, which makes them a
better carrier of this nutrient than forage silages. Fish silages can be an important protein
supplement, but their efficiency depends on the durability of their nutritional value, after
conservation by anaerobic fermentation, and the freshness of the fish byproducts used (Hussein
et al., 1991). When working with organic wastes from tilapia and other types of fish it, is of
great importance to preserve the protein, as these products are mainly suppliers there of. The
in situ method has had great acceptance among investigators and is commonly used to
estimate the degradability of feed components, like dry matter (DM) and CP (Olaisen et al.,
2003). Values determined in situ are generally good indicators of the nutritional value of feeds
preserved by anaerobic fermentation.
Objectives
Evaluate the effects of anaerobic fermentation of organic wastes from tilapia, pineapple
processing and two tropical forages; one grass and one legume, on chemical composition,
recovery of ensiled matter and DM and CP degradability.
Materials and Methods
Stargrass hay (Cynodon nlemfluensis), residues from processed pineapple (Ananas camosus),
wastes from tilapia (Oreochromis niloticus) fillet production and rhizome perennial peanut hay
(Arachis glabrata) where cut into 5.08 cm. pieces. The tilapia waste only was mixed with 30%
37

molasses. The feeds were subjected to anaerobic fermentation for a minimum of 21 days, in
PVC silos in the laboratory. Two samples of each fresh material and three samples of each
resulting silage were analyzed. The in situ method was used to determine DM and CP
degradability. Two fistulated Holstein cows, fed a grass hay diet were used. The determination
involved placing 2g of stargrass hay, pineapple organic wastes and peanut or 5g of tilapia
organic wastes in nylon bags (Dacron). Triplicate samples from each feed were incubated
intraruminally (Fig. 1,2 and 3) for 0, 3, 6, 12, 24 y 48 h, and then analyzed for DM
disappearance. After the incubation time lapsed, the bags were removed from the rumen,
rinsed in water (Fig. 4) and placed on ice for 15 min. to stop further bacterial fermentation (Fig.
5). The bags were placed in a washing machine, during one washing cycle (Fig. 6). Then the
bags were placed in a convection oven, at 65˚C for 48h to dry. Dry bags were weighed, to
determine DM disappearance. CP content from the residues at each incubation time was
determined using the Kheldajl method. This final step was needed to determine CP
degradability. Data were analyzed using the general lineal model of SAS® and Bonferroni test
of mean separation.
Results and Discussion
Table 1 presents means of DM and CP content, proportion of humid matter recovery and in situ
DM degradability of the silages. Processed pineapple silage was the lowest in DM content,
resulting from its high proportion of residual juice. Tilapia silage showed the second highest DM
content, which resulted from mixing the fish residue with sugarcane molasses. Proportion of
humid matter recovered as silage was high for all feeds, indicating a good fermentation.
CP content on processed pineapple residue was similar to that of the two tropical forage
silages. Atypically the perennial peanut was lower in CP than stargrass in this study. Tilapia
silage showed the highest CP content by a wide margen, establishing it as a potential protein
supplement in ruminant diets. Pineapple residue silage was outstanding in ruminal
degradability; tilapia silage in second place, was higher than the two tropical forages, but not
significantly so relative to perennial peanut, which had a higher DM degradability than
stargrass. Silages from these two organic wastes clearly shows a higher nutritional value in
terms of digestibility than tropical forages, which establishes them, especially processed
pineapple, as possible energy supplements in ruminant diets, whereas tilapia silage is superior
as a protein supplement.
38

Fig.1) Preparation for sample placement
Fig.2) Samples for one incubation time to be placed intraruminally
Fig.3) Intraruminal sample placement
39

Fig.4) Rinsing of all incubated samples after removal from the rumen
Fig.5) Ice bath to stop further bacterial fermentation
Fig.6) Samples in washing machine during 1 washing cycle
40

Table 1. Dry matter content (DM), recovered humid matter (RM) and ruminal
degradability of DM of two tropical forage silages and two fermented
organic wastes.
Silage DM (%) RM (%)
Crude
Protein on
dry base
(%)
Degradability
of DM at
48h (%)
Stargrass 24.285 c 97.85 ab 12.58 50.314 c
Rhizome perennial
peanut 36.11 a 99.51 a
10.41
58.383 b
Pineapple 11.181 d 96.21 bc 10.71 74.533 a
Tilapia 30.162 b 95.12 c 39.3 64.066 b
EE 1.466 1.431 14.07 0.233
Means with the same letter on the same column do not present significant differences.
DM- Dry matter
RM- Recovered matter
EE-error mean square
Fermentation characteristics of these silages are shown in Table 2. Pineapple waste silage had
the lowest pH, while tilapia silage showed a small but significant difference, relative to the two
forage silages, as did peanut silage, relative to stargrass. The higher quality of tilapia and
processed pineapple silage is indicated by their lactic acid content and also acetic acid content
in the case of pineapple silage, when compared to the legume and grass silages. Even though
there were differences in DM ruminal degradability for the various silages, no significant
difference was established between the fresh and corresponding ensiled feeds. Mean values
were 60.7% and 62.5%, respectively (Figure 7). Thus the ensiled materials mantained the
digestibilty level that were present in the fresh state.
Table 2. Fermentation characteristics of organic waste silages and silage from tropical
forages evaluated.
Sample pH Lactic acid (%)
Acetic acid
(%)
Propionic acid
(%)
Iso-butyric
Acid (%)
StargrassEstrella 5.608 a 0.59 b 0.632 ab 0.013 a 0.0008 b
Rhizome perennial
peanut 5.465 b 0.55 b 0.402 b 0 b 0 b
Pineapple 4.118 d 1.55 ab 1.334 a 0.114 b 0.021 a
Tilapia 5.306 c 2.34 a 0.366 b 0.026 b 0.006 ab
EE 0.00433 0.355 0.164 0.00102 0.000072
Means with the same letter on the same column do not present significant differences.
EE-error mean square
41

Figure 7. Effect of ensiling on the 48 hr. in situ degradability of pineapple, tilapia,
perennial peanut and stargrass.
Degradability
48h (%)
0.625
0.62
0.615
0.61
0.605
0.6
0.595
0.625
Forage Type
0.607
Fermented
Unfermented
As a result of fermentation, peanut, stragrass and tilapia increased their ruminal protein
digestibility level at 24 hr of incubation and their proportion of soluble protein (Table 3),
contrasting with pineapple, which showed opposite tendencies. The fermentation process
resulted in a reduction in the proportion of ruminal non degradable protein (RUP), in stragrass
and tilapia wastes, which were the only two feeds from which data were obtained on this
criterion.
Table 3. Determination of soluble CP fraction (CP), in situ CP digestibility at 24 hours of incubation and
ruminally un-degradable protein (RUP), of organic wastes from tilapia, pineapple and tropical forages, fresh
and ensiled.
Sample
Soluble Fraction
(%CP)
24h Digestible
incubation (%CP)
Rhizome perennial peanut
Fresh, 14.0 56.7 -
Ensiled, 46.6 63.0 -
Stargrass
Fresh, 36.3 57.8 45.6
Ensiled, 56.0 69.9 33.1
Tilapia wastes
Fresh, 21.8 52.8 54.3
Ensiled, 28.6 66.1 39.9
Pineapple waste
Fresh, 70.4 84.5 -
Ensiled, 56.7 70.6 -
42
RUP

These experimental results indicate that anaerobic fermentation is an effective method of
preserving some organic wastes, and of improving their nutritive value and ruminal digestibility.
The biggest negative effect occurred in the increase in ruminal CP degradability of silages of the
two forages and tilapia. BY contrast the CP solubility and ruminal degradability of the pineapple,
waste were higher in the fresh material than in the ensiled product. A high ruminal
degradability of protein is undesirable in feeds with a potential to supply high quality protein at
the intestinal level, as in the case of tilapia waste.
Literature Cited
Hussein,H.S and R.M. Jordan, 1991. Fish meal as a protein supplement in ruminant diets: a
review. J. Anim. Sci. 69:2147-2156.
Olaisen,V., T. Medjell, H. Volden and N. Nesse, 2003. Simplified in situ method for estimating
ruminal dry matter and protein degradability of concentrates. J. Anim. Sci. 81:520-528.
Staples,C.R., S.M. Emanuele and G.M. Prine, 1997. Intake and nutritive value of florigraze
rhizoma peanut silage for lactating dairy cows. J. Anim. Sci. 80:541-549.
43

Use of Waste Water from a Caramel Producing Plant as a Supplement in Diets of
Weaning Pigs
María del Pilar Díaz Cabán, Carmen S. Santana Nieves and Abner A. Rodríguez
Department of Animal Industry, University of Puerto Rico, Mayagϋez Campus
Abstract
Disposal of waste water represents an increase in operational costs for the caramel producing operation
in Puerto Rico and environmental pollution for the Island. Previous studies showed that it is possible to
improve weaning pigs’ performance by the addition of this waste water to the diet (Jiménez, 2003). In
this experiment, a four week trial was conducted to evaluate the addition to the diet of 0% (control) and
10% waste water from a caramel producing plant (WWCPP) on swine performance at weaning. Sixteen
Landrace shoats of 21 + 2 days of age with an average live weight of 6 kg were used in a completely
randomized design (n=4) with a boar and a gilt per pen. Feeding was on an 8% of bodyweight daily
basis. Feed intake, weight gain and feed efficiency were not affected (P>0.05) by the addition of 10%
WWCPP to the diet. Animal health status was not affected either. In the present case performance at
weaning was not improved by the dietary addition of 10% of WWCPP, as occurred in previous studies
with levels up to 7.5% WWCPP.
Resumen
Las aguas residuales de una planta productora de caramelo (ARPPC) requieren un gasto para su
disposición y son consideradas un contaminante ambiental para la Isla. Estudios previos han demostrado
que añadir estas aguas en la dieta de cerdos al destete en niveles de hasta 7.5%, mejora el consumo de
alimento, la ganancia en peso y la eficiencia de estos animales (Jiménez, 2003). En el presente estudio
dieciséis cerdos Landrace de 21±2 días de edad, con un peso vivo promedio de 6 kg, fueron distribuidos
al azar a 8 jaulas (un macho y una hembra por jaula) para evaluar su desempeño al añadir a la dieta 0 ó
10% de estas aguas durante cuatro semanas. Los cerdos fueron alimentados en base al 8% de su peso
vivo. El consumo de alimento, la ganancia en peso y la eficiencia alimenticia no fueron afectadas
significativamente con la adición de 10% de ARPPC (P>0.05). Tampoco se observaron efectos negativos
sobre la salud de los animales. Basándonos en los resultados obtenidos en este estudio podemos inferir
que la adición de 10% de ARPPC no mejoró el desempeño de cerdos al destete, diferencia de los
resultados de estudios previos con niveles menores de ARPPC.
44

Introduction
Swine producers have decreased the age at weaning in order to increase the number of pigs
weaned per sow annually and thus improve profitability (Kerr et al. 1998). The early weaned
pigs require highly digestible diets, including high quality protein ingredients, which makes the
diets more expensive (Richert et al. 1996).
In PR swine producers do not follow proper management practices for successful early weaning.
This is, in part, because of the lack of proper physical facilities and feed. The acquisition of
complex diets (for pigs under 21 days of age) represents a problem in PR because there is no
local production of this kind of diet and the only ones available are too expensive, to be
attractive to swine producers. Therefore, there is a need to identify other low-cost ingredients,
which can be easily added to the diets without compromising animal performance, and help to
reduce production costs.
On the other hand, in PR there is an environmental pollution problem due to waste disposal that
has been increasing over the years. This has stimulated interest in searching for new
alternatives that might reduce the problem. The waste waters from a caramel producing plant
(WWCPP) are considered environmental pollutants. Recent studies with the use of different
percentages of these waste waters (Léon et al., 2003; Jiménez, 2003) in the diets of weaning
pigs, have reported improved performance at the levels evaluated (up to 7.5% addition).
These previous results suggested evaluating higher levels of WWCPP in the diets of early
weaned pigs.
Objective
To evaluate the performance of weaning pigs receiving a diet containing 10% of waste water
from a caramel producing plant.
Materials and Methods
Sixteen Landrace shoats from two different litters, averaging 21±2 days of age, and 6kg body
weight, were distributed among eight pens (one male and one female per pen) at the
Department of Animal Industry Swine Farm located in Lajas, PR. Pigs were fed a commercial
weaning diet, at the rate of 8% of their bodyweight daily, with the addition of 0 or 10% of
WWCPP. These residual waters were obtained from the Coca-Cola Company plant located in
Gurabo, PR and were mixed together with the dry feed.
The pigs were weighted once a week and their feed intake was registered. The animals were
observed periodically for the presence of diarrhea or any visible change in their health status.
45

Kg
The variables feed intake, weight gain and feed efficiency were analyzed as a completely
randomized design using a lineal model by SAS statistical package (1990) with the initial
bodyweight per pen as a covariable.
Results and Discussion
The addition of 10% of WWCPP did not significantly affect (P>0.05) productive performance in
this trial (Figures 1 and 2). Feed intake and weight gain were similar between the control (0%
of WWCPP) and the supplemented (10% of WWCPP) group. The control group showed an
advantage in feed efficiency by a margen of 0.08 kg weight gain/kg intake (Figure 3), but this
difference was not significant (P>0.05). These results differ from those obtained by Jiménez
(2003), in which the WWCPP supplemented pigs showed greater feed intake, weight gain, and
feed efficiency compared with the control group (P≤0.05). However, the levels of WWCPP
evaluated in that study were lower than the level evaluated in the present trial (up to 7.5% vs.
10%, respectively).
The covariable was statistically significant (P≤0.05) for feed intake, but not for weight gain or
feed efficiency (P>0.05). Logically heavier pigs are expected to consume more feed (National
Research Council, 1987). Neither diarrhea nor any other health problems were observed in the
pigs supplemented with 10% of WWCPP.
9
8
7
6
5
4
3
2
1
0
P>0.05
P>0.05
7.7 ± .25
5.9 + .38
0 10
Levels of WWCPP (%)
7.4 + .25
Weekly Intake Weekly Gain
5.1 + .38
Fig. 1. Weekly feed consumption and weight
gain as affected by supplementation
Event with WWCPP. gain/feed
intake) was higher
46
Kg
1.2
1
0.8
0.6
0.4
0.2
0
1.10 + .04
0
.85 + .05
1.06 + .04
P>0.05 Levels of WWCPP (%)
10
Feed Intake Weight gain
.74 + .05
Fig. 2. Daily feed consumption and weight gain as
affected by supplementation with WWCPP.

Feed Efficiency
0.78
0.76
0.74
0.72
0.7
0.68
0.66
0.64
P>0.05
0.77 + .05
Conclusion
In this study no significant effects on the performance of weaning pigs resulted from
supplementation with 10% of waste water from a caramel producing plant. Feed intake, weight
gain and feed efficiency were not statistically affected as compared to the control. Although,
considering previous results, no additional benefit of increasing the level of WWCPP from 7.5 to
10% in the diet of weaning pigs was found, this industrial byproduct could be useful to
substitute for part of the imported ingredients used in swine feeding in Puerto Rico, depending
on relative costs.
0 10
Levels of WWCPP (%)
Weighing the pigs.
0.69 + .05
Fig. 3. Feed efficiency as a affected by
supplementation with WWCPP.
47
Collecting orts.

Literature Cited
Jiménez, E. 2003. Use of Waste Waters from a Caramel Producing Plant as a Supplement in the
Diet of Early Weaning and Finishing Pigs. Masters Thesis, Department of Animal Industry,
University of Puerto Rico, Mayagüez Campus. 57 pp.
Kerr C., R.D. Goodband, J.W. Smith, R.E. Musser, J.R. Bergstrom, W.B. Nessmith, M.D. Tokach
and J.L. Nelssen. 1998. Evaluation of Potato Proteins on the Growth Performance of Early-
Weaned Pigs. Journal of Animal Science. 76: 3024-3033.
León, F.J., R. Sánchez, C.S. Santana and A. Rodríguez. 2003. Waste Water from a Caramel
Industry as a Partial Substitute in Diets for Pigs. Journal of Agriculture of the Unversity of
Puerto Rico. 87 (1-2): 87-90.
National Research Council (NRC). 1987. Predicting Feed Intake of Food-Producing Animals.
National Academy Press, Washington D.C. pp. 25-41.
Richert
B.T., K.R. Cera, A.P. Schinckel, L.K. Clark and J.J. Turek. 1996. Effect of Nursery Diet
Complexity
and Weaning Age on Pig Growth Performance and Health Status. Purdue Swine
Day.
1-4.
SAS/STAT.
1990. SAS User’s Guide (Release 6.12). SAS Inst., Inc., Cary, NC.
48

The role of circulating insulin-like growth factor binding proteins (IGFBP) in the growth
of pigs
supplemented with a unique caramel wastewater (CPWW) in the diet
María de los Milagros Soltero,
Melvin Pagán, María del Pilar Díaz and Carmen Santana
University of Puerto Rico, Mayagüez Campus
Abstract
Over the past few decades, a world wide effort for the control
of industrial pollution has pinpointed
several areas on which research is needed to develop alternatives
for the disposal of polluting agents. In
the
case of the caramel industry, effective usage of the secondary waste-water products (CPWW)
generated would help to relieve this situation. Previous studies have shown that the addition of levels up
to 7.5% of this waste-water to the diet of weaning pigs improves their performance (Jimenez, 2003). At
the same time, the circulating insulin-like growth factor binding proteins (IGFBP) function to regulate the
endocrine actions of insulin-like growth factor-I (IGF-I) and recent in vitro and in vivo findings suggest
that IGFBP also
function independently of the IGF-I as growth modulators. Moreover, growth hormone
(GH) is responsible of the regulation of the overall effect of IGF and IGFBP. With these considerations in
mind,
an experiment was conducted to evaluate the expression of circulating IGFBP, IGF-I and GH in
response to a 10% inclusion of CPWW in weaning pig diets to assess associations between
those growth
related proteins and feed consumption, daily weight gain, and feed efficiency. Sixteen purebred
Landrace
pigs were randomly distributed among 8 pens (a gilt and boar per pen) and assigned one of
two treatments: 0% (control) and 10% inclusion of CPWW. During five consecutive weeks live weight
and
feed consumption were recorded. Blood samples were drawn by jugular venipuncture during the
first,
second, and third week of the experiment and serum levels of IGF-I, IGFBP-III, IGFBP-II, GH were
determined
by radioimmunoassay (RIA), immunoradiometric assay (IRMA) and enzyme-linked
immunosorbent
assay (ELISA), respectively. Serum IGF-I levels were higher in control animals (P <
0.05) and increased from d 14 to d 28 of the experimental period (P < 0.05). Weekly increases were
observed
for IGFBP-III (P < 0.05) and IGFBP-II circulating levels decreased from d 14 to d 28 of the
postweaning test period. Simple correlation analysis revealed that there was a positive association
between circulating levels of IGF-I and IGFBP-III (r = 0.88; P < 0.0001). However, the opposite was
observed between
these two and IGFBP-II (r = -0.84, P < 0.0001; r = -0.67, P < 0.0025, respectively).
Levels of GH did not show any significant change between treatments, weeks or sexes (P > 0.05). The
changes
observed in circulating levels of IGF-I, IGFBP-III and IGFBP-II may be associated with weekly
increases
in feed consumption and weight gain that occurred during the entire experimental period (P <
0.05).
Resumen
A través de las pasadas décadas, un esfuerzo a nivel mundial ha señalado varias áreas en donde se debe
realizar
investigación con el propósito de desarrollar posibles alternativas para la disposición de agentes
contaminantes.
En el caso de la industria del caramelo, el uso efectivo de las aguas residuales (CPWW)
generadas
ayudaría a aliviar esta situación. Estudios previos han demostrado que la adición de estas
aguas
de desecho hasta niveles de 7.5% a la dieta de cerdos post-destete mejora su desempeño
(Jiménez, sin publicar). Al mismo tiempo, las proteínas de enlace del factor de crecimiento
similar a
insulina (IGFBP) en el suero sanguíneo funcionan para regular las acciones endocrinas del factor I de
crecimiento parecido a la insulina (IGF-I). Descubrimientos recientes in vitro
e in vivo sugieren que los
IGFBP
también funcionan independientemente del IGF-I como moduladores del crecimiento. Mas aún, la
hormona de crecimiento (GH) es responsable de la regulación
del efecto conjunto de ambos IGF-I y
IGFBP. Con esto en mente se realizó un experimento para determinar si la inclusión de un 10% de
CPWW en la dieta de cerdos post-destete resulta en cambios en los niveles de IGFBP (II y III), IGF-I y
GH en la sangre y por ende en diferencias en crecimiento, consumo de alimento, ganancia de peso diaria
y eficiencia de conversión. Dieciséis cerdos de raza Landrace fueron distribuidos al azar a 8 jaulas (una
cerda y un cerdo por jaula) y asignados a una de dos tratamientos: 0% (control) y 10% de inclusión de
CPWW. Durante cinco semanas consecutivas se registró el peso vivo y el consumo de alimento de los
animales. Se colectaron muestras de sangre a través de sangrado yugular durante la primera, segunda y
tercera semana del experimento y los niveles de IGF-I, IGFBP-III, IGFBP-II en el suero fueron
49

determinados mediante radioimmunoensayo (RIA), ensayo immunoradiométrico (IRMA) y ensayo
inmunosorbente ligado a una enzima (ELISA), respectivamente. Los niveles de IGF-I fueron más altos
para los animales control (P < 0.05) y aumentaron del d 14 al d 28 del periodo post-destete (P < 0.05).
Se observó un aumento semanal en los niveles de IGFBP-III (P < 0.05) mientras que, los niveles de
IGFBP-II disminuyeron a partir del d 14 al d 28 (P < 0.05). Análisis de correlación simple reveló que
existe una asociación positiva entre los niveles de IGF-I y IGFBP-III circulando en la sangre (r = 0.88; P
< 0.0001). Sin embargo, se observó un efecto opuesto entre estos y IGFBP-II (r = -0.84, P < 0.0001; r
= -0.67, P < 0.0025, respectivamente). No se observó efecto de tratamiento, semana o sexo sobre los
niveles de GH (P > 0.05). Los cambios observados en los niveles sanguineos de IGF-I, IGFBP-III y
IGFBP-II pueden ser responsables de los aumentos semanales en consumo de alimento y peso vivo que
ocurrieron durante todo el periodo experimental (P < 0.05).
Introduction
Production of meat is almost entirely dependent on animal growth. However, not all aspects of
growth are understood, particularly the mechanisms involved in initiation of growth, regulation
of growth rate, and termination of growth at maturity (Hedrick et al., 1994). The Somatomedin
hypothesis originated in early efforts to understand how somatic growth was regulated by
factors secreted by the pituitary (Le Roith et al., 2001).
The hormonal regulation of growth is
multi-factorial and a group of proteins known to significantly affect animal growth and
metabolism is the insulin-like growth factor (IGF) system. The IGF system (IGF-I, IGF-II, IGF
cell surface receptors and insulin-like growth factor binding proteins) has been implicated as
being involved in many biological processes of relevance to livestock production systems.
These include pre- and postnatal growth, lactation, reproduction and the immune function. The
IGF system plays an important physiological role in the growth and development of mammals
by acting locally in specific organs or systemically through circulating IGF-I (Werner et al.,
1994). The actions of both, IGF-I and IGF–II, are mediated by specific insulin-like growth
factor binding proteins (IGFBP). The postulated modes of action of these binding proteins
include: IGF circulatory transport vehicles, retardation of IGF degradation, transvascular IGF
movement and direct modulation of IGF actions at the target cell either by enhancing or
blocking its activity (McGuire et al., 1992; Florini et al., 1996). IGFBP compete with IGF
receptors for IGF binding, and as a consequence, they can affect cell growth. The IGFBP can
act in an endocrine, paracrine, or autocrine fashion and recent in vitro and in vivo findings
suggest that IGFBP function independently of the IGF as growth modulators (Firth and Baxter,
2002). Also involved in this whole process is the growth hormone (GH). GH simultaneously
stimulates the synthesis of IGF-I and produces a microenvironment that facilitates IGF-I action
through
the modification of the IGFBP profile (Le Roith et al., 2001). Therefore, even though
great progress has occurred in meat production and
processing technologies, a basic
50

understanding of animal growth as it relates to physiology of IGFBP and their IGF independent
effects has potential for improving efficiency in meat production. A 10% inclusion of CPWW
may induce indirect changes in relative levels of IGFBP. If this inclusion causes a positive effect
on growth, it would constitute an excellent alternative for disposal of the waste water byproduct
and also benefit meat production.
Objectives
To evaluate the expression of circulating IGFBP in response to a 10% inclusion of caramel
production waste water in weaning pig diets.
To asses potential correlations between relative levels of serum IGFBP and feed efficiency, feed
consumption, weight gain and growth.
Materials and Methods
Sixteen purebred, post-weaning, Landrace pigs were randomly assigned to 8 pens (a gilt and
boar per pen) and to one of two treatments: 0% (control) and 10% inclusion of CPWW. During
five consecutive weeks live weight and feed consumption were recorded; and blood was drawn
by jugular venipuncture. Following collection, individual serum samples were diluted 1:4 or 1:1
with Laemmli’s Electrophoresis Buffer for the analysis of sample pools and individual samples
for each week. Diluted samples (2µl of serum) were subjected to SDS-PAGE
using 12%
separating acrylamide gels with 4% staking gels on a Protean II XI Electrophoresis Cell (Bio-
Rad Laboratories, Hercules, CA, USA). Prestained Protein Molecular Weight Standards (12 µl)
(Bio-Rad Laboratories) were used to estimate molecular weights of IGFBP species. Samples
were loaded on gels, control and treatment samples from each pen alternated, and all samples
were run in duplicate. Separated proteins were transferred electrophoretically onto
nitrocellulose membrane using a Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories).
Following transfer, gels were stained with Coomasie Brillant Blue R-250 (Bio-Rad Laboratories)
and membranes were stained with Ponceau S Solution (Sigma-Aldrich, Inc., St. Louis, MO, USA)
to assure a complete transfer. A biotinylated IGF-I western ligand blotting protocol was tested
for the determination of IGFBP levels
in swine serum. Membranes were washed with TBS-1%
T, blocked by incubating with TBS-0.1% T- 1% BSA, rinsed with TBS-0.1% T, and incubated
with 2 µg Biotinylated IGF-I (GroPep, North Adelaide, Australia), followed by the addition of
Strepavidin-HRP (Amersham Biosciences, Piscataway,
NJ, USA) in TBS-0.1%T. Blots were
51

insed,
treated with ECL Western Blotting Detection Reagents and Analysis System (Amersham
Biosciences) and exposed for two minutes with Kodak BioMax Light Film (Sigma-Aldrich, Inc.).
Because the validation of Biotinylated
IGF-I Western Ligand Blotting could not be achieved,
sample
pools were made and sent to Diagnostic Systems Laboratories (Webster, TX) where
they were subjected to Radioimmunoassay (RIA), to detect IGFBP-II; Enzime-Linked
Immunosorbent Assay (ELISA), to detect GH; and Immunoradiometric Assay (IRMA), to detect
IGF-I and IGFBP-III. A diagram of the pooling strategy is provided:
Days 0 14 28
Week Effect:
C + T = ♀ + ♂
Sex by Treatment Effect:
♀ ♂
C T C T
Treatment Effect:
C (all)
T (all)
Total Number of Pools = 21
1 1 1
4 4 4
2 2 2
A factorial experimental design was used to study the effect of treatment (0 and 10% CPWW),
sex (male and female), week (d 0, 14, 28 of blood collection) and the interaction between sex
and treatment (GLM Procedure; SAS, 1996). The correspondent means were separated using
the Bonferroni test and the degree of association between GH, IGF-I, IGFBP-III and IGFBP-II
was established by Pearson Correlation Coefficients (Corr Procedure; SAS, 1996).
Results and Discussion
No significant differences were found between control and CPWW supplemented pigs in weight
gain, feed consumption, and feed efficiency (P > 0.05, data not shown). On the contrary,
weekly increases in feed consumption and weight gain were observed during the entire
experimental period (Figure 1, P < 0.05). Serum IGF-I levels were higher in control animals
(Figure 2, P < 0.05) and increased from d 14 to d 28 of the experimental period (Figure 3, P <
0.05). Weekly increases were observed for IGFBP-III (Figure 4,
P < 0.05) and IGFBP-II
circulating levels decreased from d 14 to d 28 of the postweaning test period (Figure 5, P <
52

0.05). Simple correlation analysis revealed that there was a positive association between
circulating levels of IG F-I and IGFBP-III (r = 0.88; P < 0.0001). However, the opposite was
observed between these two and IGFBP-II (r = -0.84, P < 0.0001; r = -0.67, P < 0.0025,
respectively). Levels of GH did not
show any significant change between treatments, weeks or
sexes (P > 0.05, data not shown) . Unlike GH, which is secreted in a pulsatile manner, IGF-I
levels remain fairly constant throughout the day (Bach, 1999) and can be determined from a
single blood sample (Bishop
et al., 1989). At the same time, IGF-I serum concentration is
associat ed with growth traits in man y livestock species (Anderson et al., 1988; Graml et al.,
1994) and may be useful as a physiological indicator in selection programs designed to improve
weight and growth rate in pigs (Buonomo et al., 1987). IGF-I mediates many of the growth-
promoting effects of growth hormone and regulates postnatal growth and development.
However, the actions of the IGF-I and –II (IGFs) are modulated by a family of six high affinity
binding proteins (IGFBP-1–6) that have been identified by molecular cloning of their cDNAs
from rat and human tissues (Shimasaki and Ling, 1991). These proteins bind IGFs with an
affinity equal to or greater than that of the IGF-I receptor (Rechler and Clemmons,
1998) and
they
can act in an endocrine, paracrine, or autocrine fashion. IGFBP-2 and -3, like IGF-I, are
also responsive to growth hormone (Cohick et al., 1992; Harrell et al., 1999)
and may be useful
indicators of rate of gain and carcass composition in livestock. IGFBP-2 is a distinct protein
whose plasma concentration is inversely related to GH (Binoux et al., 1986) whereas IGFBP-3 is
a glycoprotein whose plasma concentration is directly related to GH secretory status (Baxter
and Martin, 1986). In the present study no association was found between serum levels of IGF-
I, IGFBP-III, IGFBP-II and GH (Table 1). This may be due to the fact that a single blood
sample was collected to measure GH instead of the recommended 10 hours sampling strategy
for accurate determination of this specific hormone. We found that circulating levels of IGFBP-
II even though decreased from day 14 to d 28 of the experimental period (Figure 5), were
higher than the levels of both IGF-I and IGFBP-III (Figure 3 and 4, respectively). Generally,
IGFBP-III is the most abundant IGFBP species in serum and milk. It is produced mainly by non-
parenchymal hepatic cells and circulates in serum, binding IGF-I or IGF-II in conjunction with
an acid-labile subunit (ALS) to form a 150 kDa circulating (ternary) complex at a serum
concentration of about 100 nM which increased the IGF-I half life to 15 h (Rechler and
Clemmons, 1998). This is in accordance with the strong positive correlation found between
these two proteins in the present study (Table 1). The precise roles of individual IGFBP are still
53

unknown, due mainly to the great complexity of their actions and their regulation, but also to
the fact that the overwhelming majority of information about the IGFBP is derived for in vitro
studies. IGFBP-2 appears to play a key role in myogenesis (Ernst et al., 1992; Ernst et al.,
1996; Fligger et a., 1998; Gerrard et al., 1999). The level of expression of IGFBP-2 mRNA and
protein was found to be high in proliferating turkey myogenic satellite cells (Ernst et al., 1996)
and mouse myoblast (Ernst et al., 1992) and to decrease gradually as differentiation
progressed. This has been associated with a sequestration of IGF-I by IGFBP-2, which makes
that growth factor less available to the myogenic satellite cells (Fligger et al., 1998). Schneider
et al. (2000) reported that the most prominent phenotype in IGFBP-2 transgenic mice was a
reduction of somatic growth and that overexpression of IGFBP-3 under a ubiquitous promoter
resulted in selective organomegaly. In our study, circulating levels of both IGFBP-III and
IGFBP-II were much higher than the levels of IGF-I. Therefore, these two binding proteins
somehow are regulating the actions of IGF-I (Table 1) and probably functioning in an IGF-I
independent manner (according to serum levels, Figures 3, 4 and 5). Therefore, the changes
observed in circulating levels of IGF-I, IGFBP-III and IGFBP-II may be associated with the
weekly increases in feed consumption and weight gain that occurred during the entire
experimental period (Figure 1, P < 0.05).
kg
12
10
8
6
4
2
0
a
1 2 3 4
Time (weeks)
Feed Consumption Weight Gain
Figure 1. Weekly feed consumption and weight gain (Kg) of post-weaning pigs.
a
b
Within each variable means lacking a common superscript letter differ (P < 0.05).
54
b
c
c
d
c

IGF-I (ng/mL)
100
90
80
70
60
50
40
30
20
10
0
a
Control Tr eatment
Figure 2. Serum IGF-I levels of controls pigs and pigs supplemented by a 10% inclusion of CPWW
during the postweaning period. Samples collected at day 0, 14 and 28 of the
experimental period wer e pooled for control
and supplemented pigs and IGF-I was
determined by Immunoradiometric assay (Diagnostic Systems Laboratories, Inc.). Within
each variable means lacking a common superscript letter differ (P < 0.05).
180
160
140
120
100
IGF-I (ng/mL)
80
60
40
20
0
a
0 14 28
Time (Days)
Figure 3. Serum IGF-I levels of postweaning pigs. Samples collected at day 0, 14 and 28 of the
experimental period were individually pooled and IGF-I was determined by
Immunoradiometric assay (Diagnostic Systems Laboratories, Inc.). Within each variable
means lacking a common superscript letter differ (P < 0.05).
55
a
b
b

300
250
200
IGFBP-III (ng/mL) 150
100
50
0
0 14 28
Time (Days)
Figure 4. Serum IGFBP-III levels of postweaning pigs. Samples collected at day 0, 14 and 28 of the
experimental period were individually pooled and IGFBP-III was determined by
Immunoradiometric assay (Diagnostic Systems Laboratories, Inc.). Within each variable
means lacking a common superscript letter differ (P < 0.05).
IGFBP-II (ng/mL)
1200
1000
800
600
400
200
0
a
0 14 28
Time (Days)
Figure 5. Serum IGFBP-II levels of postweaning pigs. Samples collected at day 0, 14 and 28 of the
experimental period were individually pooled and IGFBP-II was determined by
Radioimmunoassay (Diagnostic Systems Laboratories, Inc.). Within each variable means
b
a a
lacking a common superscript letter differ (P < 0.05).
56
c
b

Table 1. Pearson Correlation Coefficients for Serum IGF-I, IGFBP-III, IGFBP-2 and GH.
TRAIT IGF-I IGFBP-III IGFBP-II
WEEK 0.88
0.92
-0. 64
0.0001
0.0001
0.0042
IGF-I 0.88
-0.84
0.0001
0.0001
IGFBP-III -0.67
0.0025
IGF-I = Insulin-like growth factor-I
IGFBP-III = Insulin-like growth factor binding protein-III
IGFBP-II = Insulin-like growth factor binding protein-II
GH = Growth Hormone
Conclusion
Changes in circulating
levels of IGF-I, IGFBP-III, and IGFBP-II observed in control pigs and pigs
supplemented with a 10% inclusion of CPWW may be responsible of weekly increases in feed
efficiency and weight gain that occurred during a 28 d postweaning experimental
period which
reaffirms that these proteins play a key role in swine growth regulation.
Figure 6. Jugular Venipuncture of Control Pigs and Animals
Supplemented with a 10% Inclusion of CPWW in the Diet
57
GH
-0.48
0.05
-0.20
0.4145
-0.34
0.1654

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