Effect of wet feeding and enzyme supplementation on performance and immune responses of broiler chicks

Abstract

This experiment was conducted to investigate the effect of wet feeding and enzyme supplementation on performance, carcass traits, and immune responses of broiler chickens. Total of 280-day-old Ross 308 broiler chicks were used in factorial arrangement (2×2) in a completely randomized design with 4 dietary treatments and 5 replicates of 14 chickens. Dietary treatments were included of two forms of wet (1.5 kg added water to 1150 g diet) and dry diet as well as either enzyme (0.35 g/kg) or no enzyme addition to the wheat-based diet. Performance of chickens was evaluated through measurement of body weights (BWs) during day 4–44 of age, feed intake (FI), and weight gain (WG) were measured at the same period, and feed conversion ratio (FCR) was calculated. In order to carcass measurements, two birds per pen were slaughtered at the end of rearing period. Antibody titer against Newcastle and influenza viruses and sheep red blood cells was determined to evaluate the immune responses. BW, WG, and FI of broilers insignificantly increased by wet feeding (P>0.05). Heart, liver, and gizzard relative weights were significantly influenced via feeding diet in wet form (P<0.05). Digestive tract parameters include of duodenum, jejunum, and ileum weights were markedly decreased by enzyme inclusion (P<0.05). None of the immune-related parameters were statistically different among treatments. As conclusion, the results of the present trial shows that wet feeding can affect performance, moreover enzyme inclusion might influence some digestive organs of broilers.

Keywords:

• broiler chicks
• wet feeding
• enzyme
• performance
• immune responses

1. Introduction

In Iran and also some other countries around the world, wheat is the alternative of corn as a source of energy in order to alleviate the feed costs of poultry production (Nahas and Lefrançois 2001; Engburg et al. 2004). Although wheat is a suitable substitute for corn, but high proportion of wheat inclusion in diet is accompanied by viscous gel forming due to the non-starch polysaccharide (NSP) pentosans in the intestinal lumen (Annison 1991). In such situation, contact between digestive enzymes and food particles is decreased and leads to poor utilization of nutrients during digestion and absorption (Choct and Annison 1990, 1992; Van der klis et al.1993a, 1993b).

According to some researches, mixing of water with diet (wet feeding) substantially improved feed intake (FI), weight gain (WG) (Yasar and Forbes 1999), and feed conversion ratio (FCR) of broiler chickens (yalda and Forbes 1995, 1996). It has been argued that endogenous enzyme addition to the cereal-based diets helps nutrients digestion via digesta viscosity reduction (Bedford et al. 1991; Bedford and classen 1992). Moreover, enzyme inclusion in basal diet in addition to mixing diet with water supports digesta viscosity reduction and consequently performance promotion (Yalda and Forbes 1996); furthermore, wet feeding activated endogenous enzymes as well as increased the time these enzymes were active before the diet was consumed (Yasar and Forbes 2000).

Antibody plays a role in the maintenance of immune homeostasis (Bayry et al. 2005). In addition, low levels of humoral antibody may be associated to susceptibility of chickens to disease (Parmentier et al. 2004). Gao et al. (2004) reported the significantly increased anti- Newcastle disease virus (NDV) titers of chickens with NSP-degrading enzyme supplementation.

Although some researchers such as Kettunen and Rautonenand (2005) and Liu et al. (2008) reported the impact of exogenous enzyme on immunity of broiler chickens, but the literature in the field of enzyme supplementation as well as wet feeding on performance and immunity of broilers is limited. Thus, the present study was conducted to evaluate the effect of wet feeding, enzyme, and their interaction on performance and immune responses of broiler chicks.

2. Materials and methods

2.1. Birds and management

Two-hundred-and-eighty-day-old mixed sex broiler chicks (Ross 308) were obtained from a commercial hatchery, weighted on arrival and assigned to 4 treatments, and 5 replicates of 14 birds with factorial (2×2) arrangement were formulated in a completely randomized design. Broiler chickens were fed by wheat-based diets according to Table 1 during rearing period. The cultivar of consumed wheat was M18 which is a hybrid cultivar prevalent in Iran. Effect of enzyme was evaluated through 0.35 g/kg enzyme supplementation (Natuzyme plus®) to the basal diet or no enzyme added. The multienzyme constitutions were composed of xylanase, β-glucanase, cellulase, pectinase, phytase, protease, lipase, and α-amylase. Wet feeding was prepared via addition of 1.5 l of water to 1150 g dry feed or no water addition (dry feeding). Interaction of enzyme supplementation and wet feeding also was assessed. Feed and water were provided ad libitum throughout the trial. The ambient temperature was gradually decreased from 33 to 25°C on day 21 and was then kept constant.

Table 1. Ingredients and composition of the diet.

Diet composition	Starter (4–15)	Grower (15–29)	Finisher (29–44)
Wheat (10.4% CP1)	200.00	400.00	600.00
Corn	338.30	156.00	–
Soybean meal (42% CP)	391.90	370.00	321.30
Soybean oil	25.00	37.00	45.00
Calcium carbonate	17.00	14.50	13.70
Mono calcium phosphate	14.30	11.30	10.20
DL-methionine	3.30	2.20	1.80
L-lysine	1.70	1.00	–
Salt	3.50	3.00	3.00
Vitamin premix^Footnotea	2.50	2.50	2.50
Mineral premix^Footnoteb	2.50	2.50	2.50
Calculated composition
Metabolizable energy (kcal/kg)	11.97	12.35	12.56
Crude protein (%)	213.00	210.00	200.00
Lysine (%)	13.40	11.70	10.70
Methionine + cysteine (%)	10.20	8.90	8.20
Calcium (%)	9.90	8.60	8.00
Available phosphorous (%)	4.90	4.25	4.00

^a Vitamin premix provided per kg of diet: vitamin A (retinol), 2.7 mg; vitamin D3 (cholecalciferol), 0.05 mg; vitamin E (tocopheryl acetate), 18 mg; vitamin K3, 2 mg; thiamine 1.8 mg; riboflavin, 6.6 mg; pantothenic acid, 10 mg; pyridoxine, 3 mg; cyanocobalamin, 0.015 mg; niacin, 30 mg; biotin, 0.1 mg; folic acid, 1 mg; choline chloride, 250 mg; antioxidant 100 mg.

^b Mineral premix provided per kg of diet: Fe (FeSO₄.7 H₂O, 20.09% Fe), 50 mg; Mn (MnSO₄.H₂O, 32.49% Mn), 100 mg; Zn (ZnO, 80.35% Zn), 100 mg; Cu (CuSO₄.5 H₂O), 10 mg; I (KI, 58% I), 1 mg; Se (NaSeO₃, 45.56% Se), 0.2 mg. 1 Crude protein.

2.2. Performance and carcass traits

Broilers were weighted and body weight (BW) was determined throughout days 4–44 of the age. At the same time, FI and WG of overall rearing period were determined and FCR also (FI/WG) was calculated.

At 44 days of age, two broilers per pen were randomly selected, weighed, and slaughtered for carcass measurements. Carcass, abdominal fat, heart, bursa, spleen, intestine, and liver were weighed and calculated as a percentage of live BW.

2.3. Digestive organ parameters

Relative weights of digestive organs include of gizzard, pancreas, proventriculus, duodenum, jejunum, and ileum were obtained on day 44 after randomly slaughter of two chickens per pen. The length of duodenum, jejunum, and ileum also was measured and recorded.

2.4. Immune parameters

At 24 days of age, two birds per pen were randomly chosen and their blood samples were collected from brachial vein and centrifuged to obtain serum. Antibody titers against Newcastle and influenza (H2N9) viruses were measured using hemagglutination inhibition test.

Similarly at day 24 of age, two birds were randomly selected from each pen, and were inoculated via the brachial vein with 1 ml of 1% sheep red blood cells (SRBC) suspension. At day 6 after inoculation, blood samples were obtained from the brachial vein and SRBC antibody titers were measured by the microtiter procedure of Wegmann and Smithies (1966). Titers were expressed as the log₂ of the reciprocal of the highest dilution giving visible hemagglutination.

2.5. Statistical analysis

Data were subjected to ANOVA using the general linear model procedure of SAS Institute (2008) with the main effects of feed form and enzyme supplementation and the interaction between them. Means were compared using Tukey's test and were considered to be significant different at P<0.05.

3. Results

3.1. Performance and carcass traits

Regarding Table 2, however, significant effect of neither dry nor wet form of diet was observable on the whole-period performance of broiler chickens but insignificant increasing trend of BW, WG, and FI of wet fed chicks was observed (P>0.05). This improvement also found in FCR (P>0.05). Enzyme inclusion was not induced any influential impact on the whole-period performance of broilers and the trend was not consistent. Interaction of feeding method and enzyme inclusion had no noticeable impact on performance parameters.

Table 2. Effect of wet feeding and enzyme on whole-period performance of broiler chickens.

	Treatment
	FM			ES			Source of variation (significance)
Performance	Dry	Wet	SEM	Enzyme	Without enzyme	SEM	FM	ES	FM×ES
Body weight (g)
4–44	2251.00	2318.20	35.40	2263.90	2308.20	35.90	NS	NS	NS
Weight gain (g/d)
4–44	54.50	56.20	0.88	54.80	55.90	0.89	NS	NS	NS
Feed intake (g/d)
4–44	98.00	98.80	1.40	98.80	97.70	1.40	NS	NS	NS
FCR (g:g)
4–44	1.83	1.78	0.03	1.80	1.81	0.02	NS	NS	NS

Note: FCR, feed conversion ratio; FM, feeding method; ES, enzyme supplementation; NS, not significant.

As Table 3 indicates, wet feeding significantly influenced heart and liver relative weights (P<0.05) and carcass weight increased by enzyme supplementation (P<0.05). No sign of major difference in heart and liver relative weights found by enzyme supplementation. None of the experimental treatments affected abdominal fat weight. The interaction between wetting and enzyme addition was found to be significant on carcass (P<0.01) and liver weights (P<0.01).

Table 3. Carcass traits on day 44.

	Treatment
	Feeding method			Enzyme supplementation			Source of variation (significance)
Parameters	Dry	Wet	SEM	Enzyme	Without enzyme	SEM	FM	ES	FM×ES
Carcass	72.13	71.85	0.30	72.40^a	71.50^b	0.29	NS	*	**
Abdominal fat	1.41	1.48	0.09	1.41	1.47	0.09	NS	NS	NS
Heart	0.56^b	0.62^a	0.01	0.58	0.60	0.01	*	NS	NS
Liver	2.18^b	2.34^a	0.04	2.30	2.22	0.05	*	NS	**

Note: Values in the same row not sharing a common superscript differ significantly (P<0.05).

FM, feeding method; ES, enzyme supplementation;

NS, not significant; *P <0.05; **P<0.01.

3.2. Digestive organ parameters

Effects of wet feeding and enzyme supplementation on digestive organs are shown in Table 4. Results show that chickens fed by dry diet suggested greater gizzard relative weight (P<0.05). In addition, duodenum, jejunum, and ileum weights of broilers fed enzyme increased significantly compared with no enzyme supplementation (P<0.05). Different segments of intestine were not influenced by dietary treatments. Interaction of feeding form and enzyme addition were not sufficient to show any noteworthy difference.

Table 4. Digestive organs weight and intestinal length on day 44.

	Treatment
	FM			ES			Source of variation (significance)
Parameters% BW	Dry	Wet	SEM	Enzyme	Without enzyme	SEM	FM	ES	FM×ES
Gizzard	1.530^a	1.430^b	0.039	1.480	1.480	0.041	**	NS	NS
Pancreas	0.207	0.215	0.007	0.210	0.212	0.007	NS	NS	NS
Proventriculus	0.370	0.360	0.008	0.370	0.360	0.008	NS	NS	NS
Duodenum	0.650	0.640	0.016	0.620^b	0.680^a	0.016	NS	**	NS
Jejunum	1.390	1.440	0.042	1.320^b	1.500^a	0.040	NS	**	NS
Ileum	1.260	1.240	0.045	1.140^b	1.350^a	0.040	NS	**	NS
Cecum	0.600	0.650	0.023	0.640	0.620	0.023	NS	NS	NS
Length of intestine (cm)
Duodenum	35.450	35.930	0.600	34.870	36.510	0.600	NS	NS	NS
Jejunum	81.700	83.750	1.160	81.770	83.670	1.170	NS	NS	NS
Ileum	85.100	86.970	1.320	84.800	87.270	1.290	NS	NS	NS
Cecum	41.450	41.500	0.850	42.300	40.650	0.840	NS	NS	NS

Note: Values in the same row not sharing a common superscript differ significantly (P<0.05).

FM, feeding method; ES, enzyme supplementation; NS, not significant.

** P <0.01.

3.3. Immune parameters

Table 5 lists the effect of feeding methods and enzyme addition on the humoral immunity indexes of broiler chickens as well as lymphoid organs. Dietary treatments failed to induce any substantial effect on antibody titer against Newcastle, influenza, and SRBC. Spleen and bursa were not affected by dietary treatments.

Table 5. Effect of wet feeding and enzyme on humoral immunity and lymphoid organs of broiler chickens.

	FM			Treatment ES			Source of variation(significance)
Variable	Dry	Wet	SEM	Enzyme	Without enzyme	SEM	FM	ES	FM×ES
Influenza (log2)	2.800	2.900	0.210	2.900	2.900	0.220	NS	NS	NS
Newcastle (log2)	5.700	5.600	0.300	5.700	5.500	0.300	NS	NS	NS
SRBC (log2)	6.700	6.800	0.190	6.600	6.900	0.200	NS	NS	NS
Lymphoid organs (% BW)
Spleen	0.122	0.107	0.005	0.113	0.115	0.005	NS	NS	NS
Bursa of fabricius	0.068	0.060	0.004	0.064	0.065	0.004	NS	NS	NS

Note: Values in the same row not sharing a common superscript differ significantly (P<0.05).

BW, body weight; FM, feeding method; ES, enzyme supplementation; SRBC, sheep red blood cells; NS, not significant.

4. Discussion

In the current study, although wet feeding had no significant effect on BW, WG, FI, and FCR of broilers, but slightly improved these parameters compared to dry feed. Several researches have suggested that FI is increased by wet feeding (Yasar and Forbes 1999, 2000; Scott 2002). Yasar and Forbes (2000) have accounted some reasons such as increase the passing rate of digesta in gastro intestinal tract (GIT), palatability, elevated nutrient utilization, and water restriction for this FI increase. Among mentioned reasons, enhancement of digesta passing rate was confirmed by studying the intestinal morphology of chickens and it seems to be the main reason of extended FI in their research (Yasar and Forbes 2000; Scott 2002). The marginal increase of FI in the current research is in consistent with the noted experiments. However, the lack of significant difference of FI is probably due to the greater capacity of consumed wheat type to hydration rate. Scott (2002) believed that hydration rate of wheat is limiting factor in FI of broiler chickens which has diversity among the different types of wheat. Yasar and Forbes (2000) and also Scott (2002) in their studies concluded that increased FI versus constant WG, increased FCR of wet fed chicks, but in this trial the probable higher hydration of wheat type maybe reduced passage rate of digesta compared to other studies and facilitated the nutrient absorption and improved FCR of broilers to some extent. The result of this research is in contrast to Yalda and Forbes (1995, 1996).

Enzyme addition had no considerable impact on broilers performance in this experiment. Regarding Gutierrez del Alamo et al. (2008), effect of enzyme on the wheat is cultivar dependent. Physical and chemical factors, such as viscosity and hardness (Carre et al. 2002), crude protein, and starch (Pirgozliev et al. 2003) are determining factors which affect apparent metabolizable energy (AME_n) and performance of the chicken. According to Choct et al. (2004), enzyme influence especially xylanase is related to the intestinal viscosity, crude protein, and baking strength of wheat. Thereby, the current result of performance may be the consequence of wheat type and its reaction to the supplemented enzyme. The stated results are agreed with Yasar and Forbes (2000), Amerah (2008) and Gutierrez del Alamo et al. (2008) but in contrast to the Scott (2002), Kirkpinar and Acikgoz (2004), Wang et al. (2005) and Ghobadi and Karimi (2012) which observed the significant improvement of performance by enzyme inclusion.

Feeding wet diets increased heart and liver relative weights. As wet feeding led to enhanced FI and subsequently body growth, thus, increase of heart activity caused heart weight increase. It is believed that increase of growth rate which is obtained by wet feeding predisposes chicks to heart disease (Yasar and Forbes 1999). Awojobi et al. (2009) reported the significant increase of heart weight by wet feeding. Enhancement of liver weight might be related to the viscosity reduction impact of wet feeding and consequently increase of nutrient availability and metabolism in the liver. Yalda and Forbes (1996) found the same results but Awojobi et al. (2009) failed to show any significant effect in liver weight by wet feeding.

Carcass weight was increased by enzyme inclusion. Wu and Ravindran (2004) in their experiment demonstrated that xylanase supplementation had no effect on carcass recovery which is in contrast to the results of present experiment.

In the present study, giving of diet in wet form reduced empty gizzard weight significantly. Yasar (1999) by feeding of chicks with cereal grain-based foods soaked in water concluded that development of tissue glands in proventriculus, and gizzard was related to the reduced thickness of the muscular layer of these organs. Compared to dry fed chickens, lowered gizzard weight of broilers in this trial might be attributed to the effect of wet feeding to decrease the thickness of muscular layer in gizzard. The same result also was found by Afsharmanesh and Scott (2005) by wet feeding of chickens. On the contrary Awojobi et al. (2009) demonstrated the increasing trend of empty gizzard weight by increasing of added water to diet.

Relative weight of intestinal segments consists of duodenum, jejunum, and ileum substantially decreased by enzyme addition. In general, the NSP increase the viscosity of digesta, decrease the substrate and enzyme contact, and finally increase the activity of secretory mechanisms which lead to increase the size and weight of GIT. In this situation, enzymes inhibit this process by viscosity decrease of digesta (Wang et al. 2005) which expresses the reason of lowered intestinal weight. Similar finding has been demonstrated by Wang et al. (2005) and Berwal et al. (2008).

Neither wet feeding nor enzyme supplementation affected antibody titer against SRBS, influenza, and Newcastle in broilers. Lymphoid organs include of bursa and spleen weights also were not statistically influenced by treatments. According to the enhanced antibodies against NDV in response to phytase addition in the research of Liu et al. (2008) and base on the study of Gao et al. (2004), who reported the increase of anti-NDV titers of chicks by NSP-degrading enzyme supplementation, immune responses were expected to be elevated. In addition, increased bursa weight of Hubbard broilers with phytase addition also has been expressed by Zyla et al. (2000) which was not appeared in this trial. It is likely that a higher level of enzyme supplementation may be needed.

5. Conclusion

FI, BW, and FCR of broilers slightly improved by wet feeding. No consistent impact of added enzyme was observed on chicken's performance. Interaction of wet feeding and enzyme inclusion was found to be effective on the performance of chickens. Enzyme inclusion decreased relative weights of different intestinal segments which may be the result of viscosity reduction. None of the immune-related parameters were affected by either wet feeding or enzyme supplementation.