Growth Performance and Tibia Mineralization of Broiler Chickens Supplemented with a Liquid Extract of Humic Substances

ABSTRACT

The objective of the study was to evaluate the productive, carcass, and tibia mineralization responses in broiler chickens supplemented with a liquid extract of humic substances (HS) in the drinking water. Chicks were housed in holding cages from 8-42 days of age and were randomly assigned to one of five increasing HS levels in the drinking water (0, 161, 322, 483, and 644 µg/L). At 21 and 42 days, to obtain carcass and tibia measurements half of the broilers were slaughtered. ANOVA and linear regression were used to analyze the data. The HS chemical composition and flat structures were estimated. At 21 days, increasing levels of HS in the drinking water resulted in a cubic response on breast weight (p<0.05), tibia ashes percentage (p<0.05) and tibia Ca percentage, as well as a linear increasing response (p<0.05) on P percentage. HS elicited a quadratic response on the tibia DM percentage (p<0.05), Ca content (p<0.01), and P content (p<0.05) at 42 days. The optimal HS supplementation level to achieve the highest tibia DM percentage, Ca and P content were 345.00, 322.46, and 347.75 µg/L, respectively. Increasing HS levels also resulted in a cubic response in tibia Ca (p<0.05) and P percentage (p<0.01). In conclusions, HS supplementation in drinking water improved bone mineralization in broiler chickens at 21 and 42 days of age.

Keywords:
Broilers; humic substances; productive responses; tibia mineralization

INTRODUCTION

Humic substances (HS) have been studied in poultry as an alternative to substitute the growth-promoting antibiotics in order to reduce the risk of pathogenic bacteria developing antimicrobial resistance which can spread between different bacterial populations via humans, livestock, and the entire environment (Woolhouse et al., 2015). It has been demonstrated that HS improves the growth and carcass traits of broilers, as well as laying hen productivity and egg quality (Arif et al., 2019). The observed benefits of HS in poultry appear to be multifaceted, including improvements in the digestive functions, increases of beneficial bacteria in the gut, improvements in the immune response and antioxidant capacity, and thus on broiler chicken health (Arif et al., 2019; Domínguez-Negrete et al., 2019). Because HS are the most common natural complexing ligands found in nature their primary application in environmental chemistry is in the removal of toxic metals, anthropogenic organic chemicals, and other pollutants from water (Peña-Mendez et al., 2005). Because of their colloidal properties and ability to form chelates, HS have been shown to modify the toxic effects of a variety of xenobiotics and undesirable substances that enter the digestive tract in animals with feed and water (Herzig et al., 2007; Livens, 1991). Several studies have been conducted to assess the ability of HS to reduce the tissue accumulation of heavy metals such as mercury, cadmium, and zinc in fish (Hammock et al., 2003), lead and cadmium in rats (Rochus, 1983), and cadmium, zinc, and lead in chickens (Herzig et al., 2007; Zralý et al., 2008; Herzig et al., 2009).

HS are recognized in plant science by their stimulating effects on mineral uptake by the roots and mineral concentrations such as Ca, P, K, Mg, Cu, Fe, Zn, and Mn in several plant tissues (Çimrin et al., 2010; Denre et al., 2014; Canellas et al., 2015). Increases in ash and Ca content in tibia bone have also been reported in broilers fed HS (Eren et al., 2000; Disetlhe et al., 2017; Jad’uttová et al., 2019) as have increases in Ca, Fe, and Cu concentrations in broiler meat (Ozturk et al., 2010; Ozturk et al., 2012). This is consistent with the increased percentage, thickness, and hardness of eggshells reported in HS-supplemented laying hens and pheasants (Dobrzański et al., 2009; Ozturk et al., 2009).

In a previous study, supplementing drinking water with worm compost leachate as a source of HS in broilers aged 21-49 days, increased ash retention by 18-39% (Gómez-Rosales & Angeles, 2015). However, no differences in ash retention, tibia weight, and tibia ash content were observed in a later study in which a solid extract of HS (ESH) from a worm compost was supplemented in the feed of broilers from 14 to 35 days of age, (Domínguez-Negrete et al., 2019). This disparity was most likely caused by differences in the source and form of HS supplementation, the age of the chickens and the duration of HS consumption.

There are also large variations in the amounts of HS used in drinking water to boost broiler productivity. Benefits in feed conversion, energy digestibility, and ash retention were reported in one study using doses ranging from 0 to 200 µg HS/L water (Gómez-Rosales & Angeles, 2015). Improvements in body weight, feed conversion, and carcass yield were observed in another study using doses ranging from 0 to 450 mg HS/L water (Ozturk et al., 2010). As a result, in order to improve broiler production parameters, the dosages of HS added to the drinking water must be defined. Increasing doses of HS in broiler water, up to a maximum of 200 µg/L, caused a linear increase in tibia ash retention; however, it is unknown whether higher concentrations of HS in drinking water could cause additional improvements in bone mineralization (Gómez-Rosales & Angeles, 2015).

Because leg problems due to insufficient bone mineralization during the first three weeks of growth in broilers may be a limiting factor in achieving maximum performance in subsequent stages of production, the search for alternative options to improve the bone development is necessary (Díaz-Alonso et al., 2019). Therefore, the objective was to assess the productive variables, carcass traits, and tibia mineralization responses of broiler chickens supplemented with a liquid source of HS in their drinking water.

MATERIAL AND METHODS

Animals and management

The Ethical Committee of Animal Use of the National Center of Disciplinary Research in Animal Physiology, National Institute for Research in Forestry, Agriculture, and Livestock revised and approved this study in accordance with Mexican regulations (NOM-062-ZOO, 1999). A group of Ross 308 one-day-old male broilers from a regional commercial hatchery was randomly allocated in floor pens with wood shaving and gas brooder heaters. A standard broiler starter diet was provided from 1 to 5 days after hatching. On day six, 360 chicks were randomly assigned to holding cages in groups of two (30 cm wide x 38 cm deep x 37 cm height). Cages were arranged in batteries and were provided with gas heaters, equipped with plastic feeders and cup waterers. On day eight, five increasing levels of HS in the drinking water (0, 161, 322, 483, and 644 µg/L) were used. Since the maximum ash retention in broilers added with doses ranging from 161-322 µg HS /L of water was reported in previous research, but the inflection point on the ash retention was not observed, higher concentrations of HS in the water were tested in the current study. A liquid extract of HS (LEHS) was mixed into the drinking water to achieve the increasing levels of HS. The mixture of the LEHS and water was served in plastic bottles tied to the cage. The LEHS was extracted from a worm compost using an alkaline extraction procedure described previously (Domínguez-Negrete et al., 2019), and it contained 2.0% DM with 48.15 humic acid (HA) and 31.85% fulvic acid (FA). The concentrated LEHS was stored in 20 L amber plastic containers during the experiment. A starter diet from 8-14, a grower diet from 15-28, and a finisher diet from 29-42 days of age were provided. Diets were based on corn and soybean meal and were designed to meet or exceed the nutrient requirements of broilers at each stage of growth (Table 1). Diets were served ad libitum in mash form. Anticoccidial drugs and antibiotics used to promote growth were not included in the feeds.

Table 1
Ingredient composition and calculated nutrient content of the experimental diets.

	Starter	Grower	Finisher
Ground corn	63.81	65.31	67.41
Soybean meal	29.40	27.00	24.70
Vegetable oil	2.10	3.30	3.80
Calcium orthophosphate	1.70	1.65	1.52
Calcium carbonate	1.60	1.50	1.43
Salt	0.32	0.30	0.28
Sodium bicarbonate	0.20	0.20	0.20
DL-Methionine	0.24	0.21	0.18
L-Lysine·HCl	0.26	0.21	0.19
L-Threonine	0.08	0.05	0.04
Premixa	0.20	0.20	0.20
Choline chloride	0.09	0.07	0.05
Calculated nutrient content
Metabolizable energy, kcal/kg	3000	3100	3200
Digestible Lysine, %	1.10	1.00	0.90
Digestible Methionine, %	0.52	0.47	0.43
Digestible Threonine, %	0.71	0.65	0.60
Total Calcium, %	1.00	0.90	0.80
Available Phosphorus, %	0.50	0.45	0.40

• ^a
  Each kg provided: 6500 IU Vit A; 2000 IU Vit D3; 15 IU Vit E; 1.5 mg Vit K; 1.5 mg thiamine; 5 mg riboflavin; 35 mg niacin; 3.5 mg pyridoxine; 10 mg pantothenic acid; 1500 mg choline; 0.6 mg folic acid; 0.15 mg biotin; 0.15 mg Vit B12; 100.0 mg Mn; 100 mg Zn; 50 mg Fe; 10 mg Cu; 1.0 mg I.

Characterization of humic substances

The concentration of HS in the LEHS was determined as previously described (Stevenson, 1982). Also, a 5 L sample was dried in a stove at 55 °C and ground using a 2 mm mesh; the dry product was used for laboratory analysis. The functional groups were determined using an infrared spectrophotometer with Fourier transformation with attenuated total reflectance (FTIR-ATR). The elemental analysis was carried out using energy dispersive X-Ray spectroscopy (EDS). The crystal types were detected with X-ray diffraction (XRD). The results were used for the calculation of aromaticity. The data of functional groups, elemental analysis, crystal types and aromaticity percentage were used to estimate the chemical properties and the flat structures of the HS molecules with aromaticity using the chemistry software ACD Lab v.12 (Advanced Chemistry Development, Toronto, Canada).

Productive parameters

Broilers were weighed individually at the beginning, at 21 and 42 days of age, to calculate the daily weight gain (WG, g/d). Feed offered and refused was registered to calculate the daily feed intake (FI, g/d). The feed conversion ratio (FCR) was estimated by dividing the FI between the WG. At the beginning of the trial, there were 36 replicate cages per treatment with two birds per cage. At 21 days of age, half of the birds (18 replicate cages per treatment) were slaughtered by cervical dislocation to get the breast and carcass weight and yield. From 22-42 days, the rest of the birds were allocated individually but keeping the identification of the cage of origin and treatment. At 42 days, the other 18 replicates per treatment with two broilers per replicate were also slaughtered following the same procedures described before. In broilers killed at 21 and 42 days, the left and right tibias were removed; the bones from three chicks (six tibias in total) from the same treatment were pooled to ensure enough samples for the Ca and P analysis. All samples were frozen.

Laboratory analysis

The tibias were unfrozen, cleared of soft tissues, weighed and dried using a horizontal flow drying oven to estimate the DM percentage (Terlab S.A. de C.V., Zapopan, Jalisco, México) at 105 ^oC for 24 h. Then tibias were defatted in ethyl ether, and incinerated in a furnace (Furnatrol I Type 1,8200; Thermolyne, Guadalajara, Jalisco, Mexico) at 600 ^oC for 6 h to determine the ash percentage following the recommendations of Díaz-Alonso et al. (2019). The DM and ash content (g) were estimated by multiplying the DM and ash percentage by the fresh weight and dry weight of the tibia, respectively. The P percentage was determined by spectrophotometry using a GENESYS™ 10S UV Vis Spectrophotometer (Thermo Scientific, Mexico City, Mexico) and the Ca percentage was assessed using the atomic absorption methodology using an M Series AA Spectrometer (Thermo Electron Corporation, Waltham, Massachusetts, USA). The Ca and P content (mg) were estimated by multiplying the Ca and P percentage by the ash content. All laboratory determinations were carried out following standard procedures (AOAC, 2019).

Statistical analysis

Data were subjected to ANOVA using the General Linear Model of SAS (1990). Differences among means were tested by the LSD method. The growth performance and carcass traits from 8-21 and 22-42 days were analyzed using two birds per replicate and a total of 18 replicates per treatment in each period. All tibia determinations were carried out using six composite tibias from three birds for each replicate sample for a total of 12 replicates per treatment. Linear regression analysis was also used to test for linear, quadratic, and cubic responses for each response variable regarding the increasing levels of HS.

RESULTS

Characterization of humic substances

In Table 2, the chemical properties of the HA and FA molecules are presented. In Figures 1 and 2, the flat HA and FA structures with aromaticity are depicted. Higher molecular weight and higher concentration of C, H and N were estimated for HA compared to FA, while in FA a higher amount of O was observed. HA and FA had similar density.

Table 2
Estimated chemical properties of the humic and fulvic acid molecules.^a

	Humic acid	Fulvic acid
Formula	C110H105N7O50	C25H17N0O18
Molecular weight	2325.028	619.398
Elemental composition, %	C (56.82), H (4.55), N (4.22), O (34.41)	C (48.48), H (2.77), N (2.26), O (46.49)
Density, g/cm3	1.870 ± 0.10	1.935 ± 0.06

• ^a
  Using the chemistry software ACD Lab v.12.

Figure 1
Flat structure of humic acid molecule with aromaticity.

Figure 2
Flat structure of fulvic acid molecule with aromaticity.

Productive parameters and carcass traits

The growth performance variables from 8-21 and 22-42 days of age and the carcass measurements are shown in Table 3. There was not any statistical difference on the body weight at 8, 21 and 42 days of age nor on the WG, FI and FCR on the periods from 8-21 and 22-42 days among broilers supplemented with increasing levels of HS in the drinking water. The breast weight at 21 days of age showed a cubic pattern regarding the increasing levels of HS (y = 146.74 + 0.0734x - 0.0004x² - 4E-07x³; R² = 0.99; SEM = 3.069; p<0.05); the breast weight was higher in broilers supplemented with 161 and 654 µg/L of HS. The rest of the carcass measurements in 21- and 42-day old broilers were not statistically different.

Table 3
Growth performance variables from 8-21 and 22-42 days of age of broiler chickens added with increasing levels of humic substances in the drinking water.^a

	Humic substances, µg/L of water							p value c
	0	161	322	483	654	SEMb		L	Q	C
Body weight, g
8 days, g	128.2	130.2	129.9	130.7	129.3	1.249		0.48	0.23	0.99
21 days, g	719.8	734.1	738.4	734.8	730.9	8.013		0.37	0.14	0.70
42 days, g	2452.2	2503.6	2486.8	2496.8	2500.9	34.259		0.40	0.60	0.57
Period from 8-21 days
Feed intake, g/d	61.3	63.1	63.7	63.1	63.4	0.797		0.10	0.16	0.42
Weight gain, g/d	42.3	43.1	43.5	43.1	43.0	0.546		0.41	0.18	0.68
FCR	1.46	1.47	1.47	1.47	1.48	0.013		0.32	0.85	0.61
Period from 22-42 days
Feed intake, g/d	146.0	148.9	146.5	147.0	147.1	1.895		0.96	0.72	0.41
Weight gain, g/d	81.9	83.9	82.6	83.1	84.1	1.673		0.50	0.96	0.46
FCR	1.81	1.79	1.80	1.79	1.77	0.042		0.51	0.85	0.78
Carcass traits at 21 days of age
Carcass weight, g	374	385	373	364	385	56.803		0.47	0.62	0.26
Carcass yield, %	53.8	54.2	53.7	53.3	54.6	7.357		0.45	0.59	0.19
Breast weight, g	147de	151d	144de	141e	150d	3.069		0.71	0.32	0.05
Breast yield, %	21.1	21.3	20.8	20.7	21.2	0.285		0.76	0.33	0.16
Carcass traits at 42 days of age
Carcass weight, g	1324	1341	1327	1337	1359	24.491		0.40	0.71	0.58
Carcass yield, %	54.9	54.4	54.7	54.7	55.0	0.365		0.66	0.40	0.68
Breast weight, g	574	575	581	562	572	12.232		0.65	0.86	0.51
Breast yield, %	23.8	23.3	23.9	23.0	23.2	0.275		0.10	0.72	0.99

• ^a
  n = 36, using two birds per replicate.
• ^b
  SEM = estandard error of the mean.
• ^c
  L, Q and C = linear, quadratic and cubic efects.
• ^d
  ^,e Means with different superscript in the same row differ significantly (p<0.05).

Tibia measurements at 21 days of age

The tibia DM, ashes, Ca and P content in 21 days of age broilers are presented in Table 4. The increasing levels of HS in the drinking water elicited a cubic response on the tibia ashes percentage (y = 38.1577 + 0.0209x - 7E-05x² + E-08x³; R² = 0.91; SEM = 0.539; p<0.05) and the tibia Ca percentage (y = 40.02 - 0.0468x + 0.0002x2 - 3E-07x3; R² = 0.92; SEM = 1.648; p < 0.01). The percentage of tibia ashes was higher in broilers supplemented with 161 and 654 µg/L of HS (similar to the response seen on the breast weight) while the Ca percentage was higher in broilers supplemented with 322 and 483 µg/L of HS. The tibia P percentage had a linear increasing response (p<0.05) related to the increasing levels of HS (y = 14.029 + 0.0002x; R² = 0.80; SEM = 0.050). The rest of the tibia measurements were similar regardless of the levels of HS.

Table 4
Dry matter, ashes, calcium and phosphorus content of the tibia of 21 days old broiler chickens added with increasing levels of humic substances in the drinking water.^a

	Humic substances, µg/L of water							p value c
	0	161	322	483	654	SEMb		L	Q	C
Dry matter, %	35.24	36.15	35.77	35.87	36.48	0.522		0.19	0.95	0.28
Dry weight, g	2.10	1.99	2.07	2.05	2.13	0.060		0.48	0.21	0.65
Ashes, %	38.11d	40.13e	39.32de	39.18de	39.57e	0.539		0.25	0.21	0.05
Ashes, mg	802.0	797.0	814.8	804.8	845.3	28.075		0.29	0.58	0.71
Ca, %	40.20fg	36.94f	42.04g	42.20g	36.70f	1.648		0.74	0.13	0.01
Ca, mg	320.3	298.5	342.3	339.4	307.9	18.551		0.78	0.33	0.10
p, %	14.02d	14.01d	14.10de	14.15e	14.14e	0.050		0.05	0.91	0.36
p, mg	112.6	111.8	114.8	113.9	119.4	4.203		0.29	0.49	0.71

• ^a
  n = 12, using six tibias per replicate.
• ^b
  SEM = estandard error of the mean.
• ^c
  L, Q and C = linear, quadratic and cubic efects.
• ^d
  ^,e Means with different superscript in the same row differ significantly (p<0.05).
• ^f
  ^,g Means with different superscript in the same row differ significantly (p<0.01).

Tibia measurements at 42 days of age

The tibia DM, ashes, Ca and P content in 42 days of age broilers are presented in Table 5. The addition of HS resulted in a quadratic response in DM percentage (y = 42.217 + 0.00069x - 1E-05x²; R² = 0.91; SEM = 0.440; p<0.05), Ca content (y = 941.74 + 0.7739x - 0.0012x²; R² = 0.57; SEM = 45.560; p<0.01) and P content (y = 391.46 + 0.1391x - 0.0002x²; R² = 0.66; SEM = 10.187; p<0.05). Based on the derivatization of the quadratic equations, the optimum supplementation level of HS in the drinking water to achieve the maximum tibia DM percentage, Ca content and P content was estimated to be 345.00, 322.46 and 347.75 µg/L, respectively. Broilers receiving the increasing levels of HS showed a cubic response on the tibia Ca percentage (y = 34.717 - 0.0299x + 0.0002x² - 2E-07x³; R² = 0.99; SEM = 1.242; p<0.05) and tibia P percentage (y = 14.022 - 0.0002x + 7E-07x² - 8E-10x³; R² = 0.42; SEM = 0.019; p<0.01). The Ca percentage was higher in broilers given 322 and 483 µg/L of HS, while the P percentage was higher in broilers given 322 µg/L of HS.

Table 5
Dry matter, ashes, calcium and phosphorus content of the tibia in 42 days old broiler chickens added with increasing levels of humic substances in the drinking water.^a

	Humic substances, µg/L of water							p value c
	0	161	322	483	654	SEMb		L	Q	C
Dry matter, %	42.25d	43.06de	43.16de	43.35e	42.17d	0.440		0.95	0.05	0.72
Dry weight, g	7.79	8.32	8.26	8.39	8.21	0.218		0.19	0.14	0.67
Ashes, %	35.84	35.64	35.11	36.34	35.34	0.596		0.88	0.94	0.32
Ashes, mg	2785.5	2966.1	2894.4	3046.8	2891.0	73.941		0.22	0.11	0.81
Ca, %	34.73d	33.17de	35.81d	35.98d	31.11e	1.242		0.26	0.06	0.05
Ca, mg	969.0fg	984.0fg	1039.2f	1102.5f	891.9g	45.542		0.82	0.01	0.05
p, %	14.02fg	13.98f	14.04g	13.97f	13.97f	0.019		0.13	0.38	0.01
p, mg	390.6d	414.5de	409.9de	425.9e	402.0d	10.187		0.21	0.05	0.83

• ^a
  n = 12, using six tibias per replicate.
• ^b
  SEM = estandard error of the mean.
• ^c
  L, Q and C = linear, quadratic and cubic efects.
• ^d
  ^,e Means with different superscript in the same row differ significantly (p<0.05).
• ^f
  ^,g Means with different superscript in the same row differ significantly (p<0.01).

DISCUSSION

Functional groups, elemental analysis, crystal types, and aromaticity percentage results of HS have been previously reported (Domínguez-Negrete et al., 2019). The estimated higher C and N and lower O in HA compared to FA (Table 2) agree with previous reports (Steelink, 1985; Senesi & Loffredo, 1999) that analyzed HS from soils harvested in different climate regions. Figures 1 and 2 show that the more complex structure of HA, due to higher molecular weight and a greater percentage of aromaticity, compared to FA, is consistent with previous reports (Varanini & Pinton, 1995; Calace et al., 2000). While the chemical properties of composite HS (mixtures of HA and FA) from organic sources such as manure and compost have previously been reported (Ayuso et al., 1997; Rupiashi & Vidyasagar, 2009; Campitelli et al., 2012) as far as we are aware, this is the first study to publish the chemical properties and flat structures of HA and FA extracted from a worm compost. Figures 1 and 2 depict structures that correspond to other HA and FA depicted in previous reports (Buffle, 1977; Stevenson, 1982; Mirza et al., 2011). However, it has been suggested that the elemental and chemical composition of HA and FA are affected by a variety of factors, including, geographical, climatic, physical and biological conditions, as well as the chemical composition of the raw materials from which they are derived (Peña-Mendez et al., 2005).

One issue confronting animal production today is the risk of the pathogenic bacteria developing antimicrobial resistance, which can spread between different bacterial populations via humans, livestock, and the entire environment (Woolhouse et al., 2015). To deal with antimicrobial resistance, various alternatives are in place around the world, such as the ban or reduction of the use of growth promoter antibiotics in poultry, as well as an intensive search for products that can replace them, while maintaining optimal levels of growth and health of the flocks (Gómez-Rosales & Angeles, 2015; Domínguez-Negrete et al., 2019). Several products have been tested in poultry, and according to recent reports, HS appear to be a promising choice for improving broiler growth and carcass traits, as well as laying hens’ productivity and egg quality (Arif et al., 2019).

Higher lactic acid bacteria counts in the ileal content were found, but there was no effect on the total bacteria, Salmonella and Escherichia coli counts in broiler guts supplemented with HS extracted from worm compost (Maguey-González et al., 2018a; Maguey González et al., 2018b; Domínguez-Negrete et al., 2019). Previous research has found conflicting results in gut microbiology in chickens supplemented with SH. However, one of the suggested growth-promoting effects of HS is related to their ability to form protective barriers over the digestive tract´s epithelial mucosal membrane against the penetration of pathogenic bacteria or toxic substances from bacteria (Kühnert et al., 1991; Maguey-Gonzalez et al., 2018b). This effect has been linked to the macro colloidal structure of HS, which provides good shielding on stomach and gut mucous membranes (Mudronová et al., 2020). Additionally, the colloidal properties of HS and their ability to form chelates (Livens, 1991; Herzig et al., 2007) have been linked to the improved mineral uptake in plants and bone mineralization in broilers (Eren et al., 2000; Disetlhe et al., 2017; Jad’uttová et al., 2019). However, the use of an alkaline solution of HS extracted from worm compost as a means to improve bone mineralization in broilers has never been tested. That was the focus of the current research.

The lack of effects of HS on FI (Table 3) in the present research agrees with some studies (Taklimi et al., 2002; Gómez-Rosales & Angeles, 2015; Domínguez-Negrete et al., 2019) but disagrees with reports in which FI was depressed in broilers supplemented with HS (Ozturk et al., 2010; Ozturk et al., 2012). The lack of an effect of HS on the FCR contradicts previous studies in which FCR was improved in broilers fed HS (Taklimi et al., 2002; Ozturk et al., 2010; Ozturk et al., 2012). In a previous study from our laboratory, broilers given increasing amounts of worm compost leachate as the source of HS in their drinking water had lower FCR than the control birds (Gómez-Rosales & Angeles, 2015).

HS have been shown to increase broiler growth and carcass features, as well as laying hen productivity and egg quality (Arif et al., 2019). The lack of an effect of HS on carcass, breast weight, and yield in 21 and 42-day old broilers (Table 3) contradicts the increased carcass and breast weight reported in other studies in which broilers were supplemented with several sources of HS (Ozturk et al., 2010; Ozturk et al., 2012; Disetlhe et al., 2019). Furthermore, broilers fed a solid HS source derived from the same worm compost as the LEHS used in this study had higher carcass yield (Domínguez-Negrete et al., 2019).

The differences in growth and carcass characteristics observed in this study versus previous results of broiler chickens treated with HS could be attributed to a number of factors. Differences in HA and FA content, inclusion level, and form (liquid or solid) of previously used HS in broilers, as well as chain length, side-chain composition, and origin (plant, soil, peat, and coal-derived) are all important factors to consider (Pea-Mendez et al., 2005; Arif et al., 2019). In a previous study, broilers given worm compost leachate in their drinking water had higher body weight, WG, and FCR than the control birds (Gómez-Rosales & Angeles, 2015); However, in that study, the HS were collected in an aqueous solution, whereas in this study, the HS were extracted using an alkaline solution, and the HS concentration was approximately 2.6 times higher than in the first study. In more recent studies, broiler chickens treated with a solid source of HS from a worm compost had higher carcass yield (Domínguez-Negrete et al., 2019), despite the fact that the HS were added to the feed rather than the drinking water. Two additional parameters that may influence broiler responses to HS addition are the age of the birds when HS were first supplemented and the length of the HS consumption period.

Another specific issue confronting poultry production is the intensive selection of modern broilers for the optimization of growth traits, which has resulted in high incidence of leg problems due to inadequate mineralization of bones during the first three weeks of growth (Dinev, 2012; Disetlhe et al., 2017), which could be a limiting factor for achieving maximum performance in subsequent stages of production; thus, the search for alternative options to improve performance is ongoing (Díaz-Alonso et al., 2019). Supplementation of HS in broiler feed or water is another option because they are among the most common natural complexing ligands found in nature (Peña-Mendez et al., 2005) and can chelate macro- and microminerals inside the intestine improving their assimilation (Ozturk et al., 2012; Disetlhe et al., 2017; Jauttová et al., 2019). Macrominerals, such as Ca and P, are natural and essential components of tissues and body fluids, that serve a body-building function. They are thought to be the most important elements required for adequate bone development and mineralization (Díaz-Alonso et al., 2019).

It has also been proposed that HS can cause intestinal morphophysiological changes, stimulate changes in intracellular divalent calcium levels, and act as dilators, increasing mucosal and cellular permeability (Pizzari & Birkhead, 2000; Johnsson et al., 2015). Increased gut permeability, and thus cell membrane permeability in the body, may facilitate mineral assimilation and transfer from blood to bone and cells, resulting in better skeletal development (Dinev, 2012; Jad’uttová et al., 2019; Rybalka et al., 2020). These suggestions are supported by improved digestive capabilities such as increased intestinal villus height and width (Taklimi et al., 2012; Disetlhe et al., 2017; Lala et al., 2017), improved tibia ash and Ca content (Eren et al., 2000; Jad’uttová et al., 2019), and higher meat mineral concentrations, such as Ca, Fe and Cu (Ozturk et al., 2010; Ozturk et al., 2012) of broilers supplemented with HS.

The increased tibia Ca percentage at 21 and 42 days in broilers supplemented with 322 and 483 µg/L of HS, as well as the estimated higher tibia Ca content at a dose of 322.46 µg/L of HS (Tables 4 and 5) agree with the previously reported increases on tibia ash and Ca content (Eren et al., 2000; Disetlhe et al., 2017; Jad’uttová et al., 2019) and the increases in the meat concentration of Ca, Fe and Cu (Ozturk et al., 2010; Ozturk et al., 2012) in broilers supplemented with HS. These findings are consistent with the increased percentage, thickness and hardness of eggshell reported in HS-supplemented laying hens and pheasants (Hanafy & El-Sheikh, 2008; Dobrzański et al., 2009; Ozturk et al., 2009).

The current study is the first to show linear increases in P percentage in 21-day old broilers supplemented with increasing levels of HS, as well as higher P percentage and P content in 42-day old broilers supplemented with HS (322 and 347.75 µg/L of HS, respectively; Tables 4 and 5). In line with these findings, an increase in tibia P content was observed in broilers fed a diet added with canola meal and potassium humate (Disetlhe et al., 2017). However, contrary to our findings, reductions in tibia P content were observed in broilers fed HS in another study (Jad’uttová et al., 2019). This reduction could be attributed to the addition of much higher dietary amount of HS compared to the levels of HS provided in the drinking water in the current study.

HS are recognized in plant science for their stimulating effects on the growth, mineral uptake, and increases in mineral concentration of several plant tissues (Çimrin et al., 2010; Denre et al., 2014; EL-Sayed et al., 2014; Canellas et al., 2015), but it is clear that the effects of HS on the mineral use and accumulation by plant tissues vary depending on the dosage and form of application. These findings published in Plant Science, suggest that future studies with broiler chickens should define the optimum level of supplementation and the best form (liquid or solid) to supply the HS extracted from a worm compost in order to improve the efficiency of use of Ca and P and the bone mineralization responses.

CONCLUSION

The addition of increasing levels of HS in the drinking water improved the ashes, Ca and P percentages in 21-day-old broilers but the results did not allow for the calculation of an optimum supplementation dosage. In 42-day-old broilers, there were also quadratic responses in tibia DM percentage and Ca and P content with estimated optimum levels of HS supplementation of 345.0, 322.5 and 347.8 µg/L of water, respectively. The addition of HS also improved the tibia Ca and P percentages in 42-day-old broilers. The results indicate that adding a liquid source of HS to broiler chickens drinking water improved bone mineralization at 21 and 42 days of age.

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Publication Dates

• Publication in this collection
  26 Aug 2022
• Date of issue
  2022

History

• Received
  31 Sept 2021
• Accepted
  12 Nov 2021