Effect of supplementation with organic and inorganic minerals on performance, egg and sperm quality, and hatching characteristics of laying breeder hens

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1. Introduction

The effectiveness of using trace elements is an important topic in modern poultry nutrition because trace minerals are essential for normal growth and for many metabolic processes in living organisms, as they are catalysts or components of enzyme systems in most cells of the body (Swigtkiewicz et al. ., 2014), including bone and egg shell formation, egg structure and development of avian embryos (Richards, 1997), and semen quality (Barber et al., 2005).

Most mineral sources used in diets for breeding hens come from inorganic compounds such as oxides, sulfates, carbonates, and phosphates. Organic micromineral sources are an alternative to inorganic sources. Organic trace minerals do not dissociate when in a medium with acidic gastric pH, remaining neutral in terms of electrons and protected against chemical reactions with other molecules in the intestinal lumen. Consequently, there is optimization of absorption and greater bioavailability compared to inorganic sources (Swiątkiewicz et al., 2014). Chelated or complex minerals have non-metallic ligands and are organic (Vieira, 2008). Organic compounds contain a central metal atom (electron acceptor) and also contain ligands (proteins, amino acids, carbohydrates, or lipids) with at least one atom (O, N, or S) with a lone pair of electrons (Swinkels et al., 1994).

The most commonly supplied organic minerals include zinc, manganese, selenium, copper and iron. Zinc is a component of the enzyme carbonic anhydrase, which is essential for the supply of carbonate ions during eggshell formation (Robinson and King, 1963). A zinc deficiency in the breeding bird's diet results in lower egg hatching capacity, higher embryonic mortality (Kienholz et al., 1961) and lower sperm penetration into the egg (Amen and Al-Daraji, 2011). Manganese is the metallic activator of enzymes that are involved in the synthesis of mucopolysaccharides and glycoproteins and that contribute to the formation of the organic matrix of the bark. Copper plays an essential role as a cofactor for the enzyme lysyl oxidase, which is important in the formation of collagen between the bonds present in the eggshell membrane (Leeson and Summers, 2001). Iron is a component of hemoglobin and myoglobin and is involved with oxidation, reduction and electron transport, indispensable for the fundamental physiological processes of an organism (Andrews, 2002). Selenium acts on antioxidant systems, being a component of selenoproteins, and acts directly or indirectly to reduce oxidative stress (Moreira et al., 2001). Selenium is one of the most important elements in reproduction processes. A diet deficient in selenium can result in a decrease in sperm count, motility, and fertilization capacity.

Microminerals such as Zinc (Zn), Copper (Cu), Iron (Fe), Manganese (Mn), and Selenium (Se) act as catalytic or structural cofactors in enzymes and metal-containing proteins that are contained in the cells of embryos and their extraembryonic membranes. These compounds are factors that contribute to the survival of the embryo. There are specific dietary requirements for each essential trace mineral for availability during periods of growth and development, when specific trace minerals are required for the development of differentiated tissues in embryos before hatching occurs (Richards, 1997). O

The aim of this study was to evaluate the effect on performance, egg quality, variables affected by incubation and sperm quality of supplementation with chelated amino acid minerals (copper, iron, manganese and zinc) and a protein metal (selenium) in laying hen diets and roosters, formulated with inorganic minerals.

2. Materials and methods

The present study was carried out at the Aviculture Laboratory-LAVIC of the Federal University of Santa Maria (UFSM). The UFSM Ethics Committee approved all procedures used in this study. A total of 144 white Plymouth Rock laying breeder hens and 36 Rhodes Island Red roosters aged 36 to 55 weeks were used. Hens and roosters were distributed in individual cages measuring 0.33 × 0.45 × 0.40 m and 0.33 × 0.60 × 0.60 m, respectively. Hens were standardized by weight and egg production before starting the experiment. Roosters were standardized by body weight and phenotypic traits. During the period of the experiment, the hens were artificially inseminated once a week (0.05 ml semen; Rosa et al., 1995). Semen was collected from Rhode Island Red roosters fed the same diet as the hens. Food and water were provided ad libitum🇧🇷 The birds were fed every day at approximately 08:00 h. Chickens were given a daily light cycle of 16L:8D from 36 to 55 weeks of age.

2.1. Treatments

The experimental design was a completely randomized design with three treatments, eight replicates and six chickens each. A total of 12 roosters per treatment were placed in separate cages and fed the same experimental diets, where one rooster represented a replicate. The sources of organic microminerals (OMM) were: Cu, Fe, Mg and Zn chelated with amino acids. Minerals were chelated with soy feed, where the amino acid matrix used was soy feed which had a non-specific amino acid profile. Se was supplied from an exclusive yeast product with selenium in the form of metalloprotein (YesSinergy Agroindustrial Ltda.). Cu, Fe, Mg, Zn and Se in inorganic form were supplied by feeding sodium selenite (Na₂If the₃), copper sulfate (CuSO₄), iron sulfate (FeSO₄), manganese oxide (MnO) and zinc oxide (ZnO) from rocky sources. Breeder hens were fed a basal diet based on soy and corn chow at 35 weeks of age. At 36 weeks, three experimental diets were assigned to roosters and hens: basal diet (BD) containing only inorganic mineral sources (10 mg of Cu, 60 mg of Fe, 70 mg of Mn, 75 mg of Zn and 0.3 mg of Se per kg of diet), as described in Table 1. The BD was composed of 500 g of organic minerals per ton of diet (BD +500 g OMM) elaborated with the addition of 2.5 mg of Cu, 17.5 mg of Fe, 20 mg of Mn, 27.5 mg Zn and 0.08 mg Se/kg diet, and there was a BD to which 800 g OMM was added per ton of diet (BD +800 g OMM), including 4 mg Cu, 28 mg Fe , 32 mg of Mn, 44 mg of Zn and 0.128 mg of Se per kg of diet. The BD, composed of all ingredients and inorganic minerals, was mixed in a horizontal mixer and the experimental diets were mixed in another horizontal mixer, with the addition of previously determined amounts of organic minerals. For each diet, the blenders were cleaned to avoid contamination with ingredients that should not be included in the diet.

2.2. Hen performance and egg quality

Eggs were collected and numbers were recorded four times a day. Egg production by each hen was calculated weekly. Daily feed intake (g/hen per d), feed conversion (kg feed/dozen eggs produced and kg feed/kg eggs produced) and body weight (BW) were calculated at 28-d intervals.

Egg weight, yolk weight, albumen weight, egg shell weight and specific gravity were determined weekly, totaling 20 analyzes during the experiment period. A total of 24 eggs from each treatment group (three eggs for each of eight replicates) were used in these analyses. Egg weight, yolk weight and albumen weight were determined using a precision balance (0.001 g). The eggs identified as

Table 1

Basal diet composition and nutritional contents of experimental diets.

Ingredients	DB	BD+500gOMM	BD+800gOMM
Corn (g/kg)	650.22	650.22	650.22
Soy feed (460 g/kg protein) (g/kg)	241.73	241.73	241.73
Soybean oil (g/kg)	1.49	1.49	1.49
Dicalcium phosphate (180 g/kg P; 210 g/kg Ca) (g/kg)	10.66	10.66	10.66
Limestone (380 g/kg Ca) (g/kg)	81.88	81.88	81.88
Salt (g/kg)	4.00	4.00	4.00
Premix of inorganic vitamins and minerals1 (g/kg) Organic minerals2	10.0	10.0	10.0
Chelated Copper Amino Acid (mg/kg)	–	2.5	4
Chelated Manganese Amino Acid (mg/kg)	–	20	32
Zinc Amino Acid Chelate (mg/kg)	–	27.5	44
Chelated Iron Amino Acid (mg/kg)	–	17.5	28
Selenium yeast (mg/kg)	–	0.08	0.128
Iodine (mg/kg)3 Calculated Nutritional Composition (g/kg)	–	0.6	0.96
Crude Protein	17.00	17.00	17.00
Metabolizable energy (kcal/kg)	2768	2768	2768
Calcium	35.0	35.0	35.0
Available Phosphorus	3.0	3.0	3.0
Analysis of nutritional composition (g/kg)
Crude Protein	16.58	15.49	15.60
Gross energy (kcal/kg)	3893	3958	3884
ashes	14.38	13.29	15.69
Calcium	33.5	33.4	33.3
Total Phosphorus (Total P)	5.1	5.1	5.1

¹Mineral and Vitamin Premix: Levels per Kg of Diet (DSM Nutritional Products Ltd.): Vitamin A 1500 IU; vitamin D3 4500 IU; vitamin E 80 IU; vitamin K3 5 mg; vitamin B1 3.5 mg; vitamin B2 12 mg; vitamin B6 6 mg; vitamin B12 40 mcg; niacin 60 mg; pantothenic acid 20 mg; biotin 0.4 mg; folic acid 3 mg; iron 60 mg; copper 10 mg; zinc 75 mg; manganese 70 mg; ascorbic acid 0.15 mg; selenium 0.3 mg; iodine 10 mg; methionine 1.04 g; phytase 600 FYT/g; cobalt 1 mg; enramycin 5 mg; lysine 0.15 g; Protease 11250 Prot/kg.

• YesSinergy Brasil (Yes Minerals) 360 – Birds.
• Inorganic Iodine (Yes Minerals) 360– Poultry.

Intact eggs were subjected to an evaluation of specific gravity using the method of immersion of eggs in a saline solution. Seven solutions, with densities between 1070 and 1100 g/cm³, with gradual variations of 0.05 between the solutions, were prepared. Specific gravities were determined using a densitometer and Archimedes' Principle as described by Peebles and McDaniel (2004). After completion of specific weight assessments, samples of three eggs per experimental unit were used to determine albumen height.

Measurements in millimeters (mm) were associated with egg weight, thus determining the Haugh unit: 100* log (H − 1.7 W0.37 + 7.6)

H = where albumen height (mm) and W = egg weight (g).

Yolk quality was assessed by measuring bud height (YH) and bud width (YW), and bud index (YI) was calculated as the ratio of these parameters as YI = YH/YW. The three yolks and three albumen samples were mixed separately before determining the pH using a digital benchtop pH meter.

The eggshells from the previous analysis were used to determine the thickness and weight of the shells. The weight and thickness of the peels, however, were determined every 28 days (totaling five analyzes during the entire period of the experiment). The eggshell was weighed after being dried at room temperature for 72 h (RodriguezNavarro et al., 2002). Shells from three eggs per experimental unit were used to determine shell thickness with a 0.001 mm outer electronic micrometer at three points in the equatorial zone of each egg (Lin et al., 2004). Eggshell strength was determined using a TA.XT2 Texture Analyzer texturometer with a 5 mm stainless steel Cyln probe (Texture Technologies Corp. and Stable Micro Systems Ltd., Hamilton, MA) on three additional eggs per replicate.

2.3. Rooster performance and sperm quality

Daily feed intake (g/male per d) and body weight (BW) were calculated every 28 days. Semen was collected fortnightly using the dorsal and abdominal massage method, in graduated Falcon tubes placed in a water bath at 37°C. After collection, ejaculate volume, sperm motility, vigor and sperm pH were checked. Sperm motility was determined as the percentage of motile spermatozoa (in a straight and progressive manner), and the vigor of sperm motility was determined based on motility characteristics (Celeghini et al., 2001). For this analysis, 5 μL of semen was deposited on a heated slide and observed using optical microscopy, with a magnification of 200x. fresh semen pH value was determined using test strips (MColorpHastTM; Merck Millipore, Billerica, MA, USA). For sperm concentration and morphology analysis, 5 μL of semen were added to 5 ml of formalin:citrate solution. To determine the

sperm concentration, the semen was diluted at a ratio of 1:1000, and the sperm cell count was performed using a hemocytometer (Neubauer Chamber) with a result expressed in number of cells per mm³ of semen according to the technique described by Brillard and McDaniel (1985). The result was transformed into the number of cells per ml of semen. A total of 100 spermatozoa were evaluated using oil immersion using a phase contrast microscope (1000x magnification), and the spermatozoa were classified as having normal or abnormal structures (categorized into head, midpiece and tail abnormality), and the total percentage of normal or abnormal spermatozoa was calculated.

2.4. Incubation variables

To evaluate hatchability, hatchability of fertile eggs, fertility, embryonic mortality, punctured eggs, chick quality and weight, eggs were incubated every week (total of 20 incubations). Only healthy eggs with no visible abnormalities were used for incubation. All eggs collected daily were classified and separated for incubation for each experimental group. Those eggs considered suitable for hatching were stored for a maximum period of 7 days in a temperature-controlled room (18–20° C and 75%–80% RH (relative humidity)). Incubation was performed in a commercial multistage incubator (Casp, Amparo, SP, Brazil) at 37.5° C and 60% RH. At 18 days, the eggs were transferred to the incubation equipment, which was calibrated at 36.5°C and 65% RH. The incubation rate was determined in relation to the total number of eggs incubated. Fertility refers to the percentage of hatched eggs that were fertile, while hatchability is the percentage of fertile eggs that hatched. Chicks were removed from the incubator, weighed and classified into first and second quality chicks. Chicks were considered second quality when there was an umbilicus, beak abnormalities, lower limb weakness or drooping and excessively wet feathers. To assess the hatching rate, fertility and embryonic mortality of fertile eggs, the eggs from which

incubation did not occur were submitted to embryonic diagnostics. In this evaluation, the eggs were classified, using macroscopic visual examinations, as infertile or as: Embryo mortality occurred during the first 48 h of the incubation period (EM1); Embryo mortality occurring between the 3rd and 7th day of the incubation period (EM2), Embryo mortality occurring between the 8th and 14th day of the incubation period (EM3); Embryo mortality occurred during the 15th and 21st days of the incubation period (EM4); and perforated eggs – those in which the embryos have broken the shell, but from which embryos have not emerged at the time of removing the chicks from the incubation equipment but with the embryos still alive.

2.5. Experimental design and static analysis

The experimental design was a completely randomized design with three treatments of eight replicates performed with six chickens. Fed the same diets as the hens, 12 roosters were used per treatment, where one rooster represented a repeat. Each rooster represented a repetition. All data were subjected to an analysis of variance. Each variable was assessed for normality and variance heterogeneity before performing the ANOVA. When there were significant differences in P < 0.05 Tukey's Test was used for comparison between treatments. Statistical procedures were performed using the SAS Institute (2016).

3. Results

3.1. Hen performance and egg quality

Data on egg production are included in Table 2. Egg production was higher in the BD +800 g OMM treatment group than in the control (BD) group at weeks 43, 44, 45 and 49 (P = 0.0275, P = 0.0065, P = 0.0112 and P = 0.0285, respectively), and during the other weeks there was a trend towards higher egg production from hens fed BD 800 g OMM compared to BD. This trend was also observed for the total period average with hens fed BD + 800 g OMM (79.72%) having a higher average egg production than hens fed BD (75.68%). BW and feed intake were not affected by dietary inclusion of organic microminerals (P > 0.05; data not shown). Feed conversion kg/kg (BD, 2.59; BD + 500 g OMM, 2.45 and BD + 800 g OMM, 2.51) and kg/d (BD, 1.86; BD + 500 g OMM, 1.74 and BD + 800 g OMM, 1.76 ) were not affected due to the inclusion of OMM in the diets

(P > 0.05; data not shown). Weight of eggs (P = 0.2863), yolk weight (P = 0.9634), albumen weight (P = 0.1692), eggshell weight (P = 0.3337), specific gravity (P = 0.3731), Haugh unit (P = 0.9581), yolk pH (P = 0.6171), albumen pH (P = 0.6989), eggshell thickness (P = 0.5150) and shell strength (P = 0.7979) were not affected due to the inclusion of OMM in the diets (Table 3). The yolk index was higher in eggs (P = 0.0092) from chickens fed BD than in eggs from chickens fed BD + 800 g OMM (Table 3).

3.2. Rooster performance and sperm quality

Supplementation with organic minerals did not affect BW and feed intake among roosters (P > 0.05; data not shown). Data for rooster sperm quality are included in Table 4. Semen volume (P = 0.1054), sperm motility (P = 0.4608), sperm concentration (P = 0.7550), total number of abnormal sperm (P = 0.4650), abnormality in the structure of the sperm head (P = 0.4650), abnormality in the structure of the sperm midpiece (P = 0.6421), and abnormality in sperm tail structure (P = 0.3174) were not affected by the inclusion of OMM in the diet. The semen pH values (P = 0.6402) ranged from 7.0 to 8.0, with no differences between treatments. There was no effect of organic mineral supplementation on semen pH. Sperm vigor was higher (P = 0.0262) for roosters fed BD + 800 g OMM than among roosters fed BD.

Table 2

Egg production of laying hens fed diets containing organic microminerals¹.

Age (weeks)	Treatments
	DB	BD+500gOMM	BD+800gOMM	WITHOUT*	P value
36	75.59 ± 2.17	74.13 ± 2.17	77.38 ± 2.17	6.15	0.1370
37	75.00 ± 1.51	77.38 ± 1.51	75.85 ± 1.61	4.28	0.5418
38	82.73 ± 1.94	83.63 ± 1.94	87.50 ± 1.94	5.49	0.2072
39	82.14 ± 2.26	80.35 ± 2.26	86.60 ± 2.26	6.39	0.1565
40	80.19 ± 3.05	81.94 ± 3.05	83.03 ± 3.05	8.65	0.8043
41	78.87 ± 2.49	80.65 ± ± 2.49	85.41 ± ± 2.49	7.06	0.1838
42	80.65 ± 3.08	83.92 ± 3.08	80.65 ± 3.08	8.71	0.6910
43	76.48 B ± 1.87	77.21 ab ± 2.00	83.63 a ± 1.87	5.30	0.0275
44	77.21 B ± 1.64	77.67 B ± 1.53	84.69 a ± 1.64	4.34	0.0065
45	71.72 B ± 2.51	75.89 ab ± 2.51	84.01 a ± 2.69	7.12	0.0112
46	77.21 ± 2.94	77.38 ± 2.75	78.57 ± 2.75	7.78	0.9321
47	74.40 ± 3.35	76.48 ± 3.35	75.26 ± 3.35	9.49	0.9077
48	75.59 ± 3.04	75.59 ± 3.04	82.27 ± 3.04	8.62	0.2254
49	72.02 B ± 2.47	77.97 ab ± 2.47	82.14 a ± 2.47	6.98	0.0285
50	73.47 ± 3.07	77.38 ± 2.87	76.19 ± 2.87	8.12	0.6449
51	72.91 ± 1.71	74.70 ± 1.71	77.97 ± 1.71	4.85	0.1317
52	72.79 ± 2.58	77.67 ± 2.42	77.68 ± 2.42	6.84	0.3104
53	77.21 ± 2.64	74.10 ± 2.47	73.51 ± 2.47	7.01	0.5653
54	71.27 ± 2.75	73.80 ± 2.75	73.80 ± 2.75	7.79	0.7581
55	66.96 ± 3.15	72.91 ± 3.15	72.91 ± 3.15	8.91	0.3245
Medium (36–55 weeks)	75.72 ± 1.53	77.53 ± 1.53	79.95 ± 1.53	4.33	0.1976

^{The – B} The averages in a row, without sharing a common index, are different (P ≤ 0.05).

¹ Data represent the means of eight replicates (ie, compartments) per treatment; *WITHOUT Aggregate, n = 8.

Table 3

Values for variables associated with egg quality from 36 to 55 week old hens¹.

Variables	Treatments
	DB	BD+500gOMM	BD+800gOMM	WITHOUT*	P values
Weight of eggs (g)	59.95 ± 0.55	59.22 ± 0.59	58.67 ± 0.55	1.57	0.2863
Yolk weight (g)	16.69 ± 0.16	16.72 ± 0.17	16.66 ± 0.16	0.47	0.9634
Albumen weight (g)	38.03 ± 0.40	37.24 ± 0.43	36.94 ± 0.40	1.13	0.1692
Weight of eggshells (g)	5.22 ± 0.07	5.24 ± 0.07	5.10 ± 0.07	0.20	0.3337
Specific gravity (g/cm³)	1083.9 ± 0.52	1084.8 ± 0.56	1083.7 ± 0.52	1.49	0.3731
haugh unit	95.01 ± 0.42	94.92 ± 0.45	94.84 ± 0.42	1.19	0.9581
gem index	0.443 a ± 0.00	0.441 ab ± 0.00	0.436 B ± 0.00	0.93	0.0092
yolk pH	5.92 ± 0.01	5.94 ± 0.01	5.93 ± 0.01	0.03	0.6171
albumen pH	8.34 ± 0.03	8.30 ± 0.03	8.31 ± 0.03	0.09	0.6989
Thickness of eggshells (mm)	0.373 ± 3.67	0.377 ± 3.93	0.371 ± 3.67	10.40	0.5150
Shell strength (N)	35.79 ± 1.09	35.07 ± 1.16	34.78 ± 1.09	3.08	0.7979

^{The – B} The averages in a row, without sharing a common index, are different (P ≤ 0.05).

1 Data represent means of eight replicates (ie, compartments) per treatment; *WITHOUT Aggregate, n = 8.

3.3. Effects on variables associated with embryonic and egg development during incubation

Hatching capacity (P = 0.3527), incubation capacity of fertile eggs (P = 0.0750), total embryonic mortality (P = 0.7347) in EM1 (P = 0.8002), EM2 (P = 0.3548), EM3 (P = 0.3548) and EM4 (P = 0.5959) were not affected by the inclusion of OMM in the diets. Also, the number of perforated eggs (P = 0.4929), number of second quality chicks (P = 0.6450) and chick weight (P = 0.2866) were not affected by the inclusion of OMM in the diets. Fertility was higher (P = 0.0130) among chickens in the groups that were fed BD + 500 g OMM and BD + 800 g OMM than among chickens fed BD. (Table 5).

4. Discussion

4.1. Variables associated with hens and egg quality

Different micromineral supplements did not affect the values of variables associated with performance, except for egg production. The findings that egg production was higher when BD +800 g OMM was fed than BD during weeks 43, 44, 45 and 49 of age, and that there was a trend towards higher egg production during the other weeks when the diet was supplemented with OMM are inconsistent with results for egg production from a previous study (Carvalho et al., 2015). In this previous study, there was no

Table 4

Rooster sperm quality during the experiment period (36–55 weeks of age)¹.

Variables	Treatments
	DB	BD+500gOMM	BD+800gOMM	WITHOUT*	P values
Volume (mL)	1.21 ± 0.08	0.96 ± 0.08	0.98 ± 0.08	0.30	0.1054
Motility (%)	89.30 ± 1.09	89.13 ± 1.09	90.90 ± 1.09	3.79	0.4608
Force2	3.91 B ± 0.08	4.16 ab ± 0.08	4.21 a ± 0.08	0.27	0.0262
pH	7.98 ± 0.02	7.98 ± 0.02	7.95 ± 0.02	0.07	0.6402
Concentration3	3.89 ± 0.19	3.74 ± 0.19	3.69 ± 0.19	0.67	0.7550
TAS (%)	4.97 ± 0.51	4.24 ± 0.51	5.08 ± 0.51	1.79	0.4650
AH (%)	2.21 ± 0.24	1.98 ± 0.24	2.38 ± 0.24	0.84	0.5212
AIP (%)	2.35 ± 0.25	2.08 ± 0.25	2.41 ± 0.25	0.89	0.6421
AT (%)	0.31 ± 0.05	0.30 ± 0.05	0.21 ± 0.05	0.18	0.3174

^{The – B} The averages in a row, without sharing a common index, are different (P ≤ 0.05). ¹ Data represent the means of 12 replicates (ie, compartments) per treatment; *WITHOUT Aggregate, n = 12. ²Sperm Vigor (scores of 1–5).

³ Sperm concentration (number of cells x 10⁹ mL of semen).

TAS = Total sperm abnormality, AH = head abnormality, AIP = Midpiece abnormality, AT = tail abnormality.

Table 5

Reproductive responses (%) evaluated during the period of the experiment with 36–55 week old laying breeder hens¹.

Variables	Treatments
	DB	BD+500gOMM	BD+800gOMM	WITHOUT*	P values
incubation capacity	84.47 ± 1.63	85.15 ± 1.63	87.72 ± 1.63	4.63	0.3527
Hatching ability of fertile eggs	92.64 ± 0.92	89.94 ± 0.92	92.73 ± 0.92	2.61	0.0750
Fertility	90.98 B ± 1.05	95.08 a ± 1.05	95.54 a ± 1.13	2.99	0.0130
Total embryonic mortality	5.62 ± 0.81	6.53 ± 0.81	6.02 ± 0.81	2.30	0.7347
EM12	1.54 ± 0.34	1.56 ± 0.34	1.83 ± 0.34	0.98	0.8002
EM23	1.06 ± 0.26	1.39 ± 0.26	0.84 ± 0.26	0.75	0.3548
EM34	1.12 ± 0.22	1.24 ± 0.22	0.84 ± 0.22	0.65	0.4723
EM45	1.89 ± 0.42	2.33 ± 0.42	2.49 ± 0.42	1,21	0.5959
perforated	1.27 ± 0.33	1.63 ± 0.33	1.06 ± 0.33	0.95	0.4929
second quality chicks	1.60 ± 0.35	1.27 ± 0.35	1.74 ± 0.35	1.01	0.6450
Weight of chicks (g)	41.46 ± 0.35	41.55 ± 0.35	40.79 ± 0.35	1.00	0.2866

^{The – B} The averages in a row, without sharing a common index, are different (P ≤ 0.05).

¹ Data represent the means of eight replicates (ie, compartments) per treatment; *WITHOUT Aggregate. n = 8, Incubations per week, total: 20.

²EM1 = embryonic mortality in the first 48 h of incubation.

³EM2 = embryonic mortality occurred between d 3 and 7 of incubation.

⁴EM3 = embryonic mortality occurred between d 8 and 14 of incubation.

⁵EM4 = embryonic mortality occurred between d 15 and 21 of incubation.

effect on egg production of laying hens when a mixture of organic trace minerals (Cu, Fe and Mn chelated with amino acids and partially hydrolyzed proteins) was included in the basal diet replacing 100%, 90%, 80%, or 70% of the inorganic minerals.

Kirchgessner and Grassmann (1970) reported that organic minerals form stable complexes, which decrease the possibility of forming precipitated salts with compounds such as phytic acid or insoluble fibers.

Organic microminerals, therefore, are more available for biological functions due to greater solubility and absorption when in organic form, with a facilitation of these processes by the organic binding components. In the present study, there was an increase in egg production with the use of organic microminerals, which may be related to these positive characteristics of the OMM from the perspective of solubility and absorption. The other variables associated with performance were unaffected, which could be expected because the basal diet used for all treatments was already balanced to meet the hens' requirements for these micronutrients.

Egg quality was not affected by dietary supplementation with OMM. This finding is consistent with the results of a previous study (Stefanello et al., 2014) in which an assessment of egg quality was performed. In the previous study, laying hens between 47 to 62 weeks of age were fed diets supplemented with an organic source (proteinates) of trace minerals (Mn, Zn, and Cu), and there was no effect on egg specific gravity. Also, Saldanha et al. (2009) evaluated the effect of

supplementation with organic minerals (Zn, Fe, Mn, Cu, I, and Se). In this previous study, there was no effect of these supplements on egg quality of laying hens (83 weeks of age), and there was no effect of treatments on yolk and albumen percentages. In that previous study, however, there was an effect on the specific gravity of eggs and on the percentage of shells when 80% of organic microminerals was included to replace the inorganic minerals. Garcia et al. (2010) reported that an alkaline pH negatively affects the vitelline membrane. Also, alkaline ions in albumin, such as Na, K, and Mg, can be transported from the albumen to the yolk. The migration of alkaline ions can lead to a rearrangement with the

hydrogen ions present in the yolk, which could lead to an increase in yolk pH. This pH change could lead to a denaturation of the protein in the yolk, increasing the yolk viscosity. In the present study, the inclusion of OMM in the hen diets did not affect the pH of the yolk and albumen, therefore, there should not be problems of this type if there were the inclusion of OMM in the diets of laying hens. Very little research has been carried out with breeding birds used for the production of laying hens, which makes it difficult to compare results between studies.

Sources of organic minerals used individually or together did not affect values for Haugh units (Saldanha et al., 2009; Yenice et al., 2015) or gem index (Saldanha et al., 2009). In the present study, the values for the Haugh unit were not affected by the addition of organic minerals in the diet, but the yolk index was higher among birds fed BD than BD +800 g of OMM. As eggs deteriorate, the yolk index score becomes lower because the fiber structure of the vitelline membrane loosens and the strength of the membrane decreases (Fromm, 1967). In the present study, egg yolk index values among hens fed BD (inorganic minerals only) were greater than yolk index values among hens fed BD +800 g OMM; that is, the buds showed a greater membrane fiber structure. Still, values for yolk indices for all treatment groups remained within what is considered appropriate for eggs from laying hens, which would be between 0.3 and 0.5 (Yannakopoulos and Tservenigousi, 1986).

In the present study, there was no difference in the thickness and resistance of the eggshells due to the treatments that were imposed. These findings of the present study are consistent with those of Mabe et al. (2003), where evaluations of dietary supplementation with Zn, Cu, and Mn were performed among laying hens, and there was no effect on eggshell quality: shell percentage, or shell index. In that previous study, there was greater resistance to egg breaking and fracture resilience, which is inconsistent with the findings of the present study. Swiatkiewicz and Koreleski (2008) evaluated the addition of Zn and Mn from organic and inorganic sources among laying hens between 35 and 70 weeks of age, and reported that there was no change in the percentage and thickness of eggshells.

The observed effects on the mechanical properties of eggshells indicate that microelements can interact directly during calcium carbonate formation processes by affecting the texture of the shell. The presence of microelements alters the initial phase of shell formation (Mabe et al., 2003). Bain (1990) investigated the relationship between fracture resilience and the ultrastructural organization of the shell, and suggested that supplementation with microelements promotes early melting during the initial stages of shell formation and thus improves the mechanical strength of the egg regardless of its thickness. This effect may explain the absence of differences in terms of weight and thickness of the peels observed in the present study.

4.2. Effects on roosters and sperm quality

Sperm volume in roosters was not different between treatment groups. Shan et al. (2017), however, evaluated the effect of a premix of inorganic and organic microminerals (Zn, Mn, Cu, Fe and Se) on semen quality among male broilers for rearing at 31 to 35 weeks, and concluded that there was an increase in values for variables associated with semen, such as volume and density, among roosters fed organic minerals. Organic minerals (Cu, Zn, Mn and Se) were provided by Mahan et al. (2002) and added to the diets to assess boar fertility, and the results indicated that the number of semen doses that could be used for artificial insemination by ejaculation increased from 10.9 to 23.4. Barber et al. (2005) suggested, based on research results with breeding roosters, that microminerals (Se, Mn, and Zn) exert functions on reproductive tissues during spermatogenesis to improve semen quality.

According to Surai et al. (1998), Se supplementation affects the antioxidant status of rooster semen. Edens (2002) reported that when chickens were fed a basal diet containing 0.28 ppm of inorganic Se, the percentage of normal sperm was only 57.9%, with two important abnormalities, curved midpieces (18.7%) and corkscrew heads. (15.4%). When organic Se, however, was included in the diet of broilers in the same amount, semen quality improved even more and these abnormalities decreased to 0.7% and 0.2%, and the percentage of normal sperm increased to 98.7%. The results of these studies indicate that the inclusion of selenium in poultry diets results in increased sperm counts, and using an organic source leads to a reduction in the percentage of defective sperm, thus having a positive effect on the fertilizing capacity of males. In the present study, sperm concentration and abnormalities between spermatozoa were the same when organic and inorganic minerals were fed. Notwithstanding this, the results of the present study are consistent with the results of the previous studies described above. Although there was no effect on the values for these variables, sperm vigor was greater among males fed BD +800 g OMM than among those fed BD without inclusion of OMM. Furthermore, the fertility of incubated eggs was higher when OMM was included in the hen diets (discussed subsequently in this manuscript).

The increase in sperm cell vigor may be related to the results reported by Renema (2004). In this previous study, breeder hens fed organic Se had a higher number of sperm holes at the point of fertilization in the perivitelline membrane compared to hens fed inorganic Se. This effect was attributed to changes in the oviductal milieu, such as a reduction in free radicals in sperm host glands, due to increased glutathione peroxidase (GSH-Px) activity.

Other trace minerals relevant to sperm quality are Mn, Cu and Zn. Mn and Cu are powerful stimulators of sperm motility (Lapointe et al., 1996). Amen and Al-Daraji (2011) reported that zinc is important for cell division and the production of viable sperm, and it is the most important micromineral for the reproductive functions of male animals. Testosterone metabolism is necessary for testicular growth, sperm production, and sperm motility. In this previous study (Amen and AlDaraji, 2011), an evaluation of the effect of dietary supplementation with different concentrations of Zn on broiler hens was evaluated, and there was a greater penetration of eggs by sperm compared to when there was no Zn supplementation. Shan et al. (2017) reported that there was an increase in sperm motility and the number of normal sperm among breeding roosters fed an organic micromineral premix than among roosters fed an inorganic mineral premix at 31 to 35 weeks of age. In the present study, sperm motility and the number of abnormal spermatozoa were not affected by feeding with organic minerals. Further research on sperm quality among roosters could be carried out to elucidate the effects of using minerals in organic form.

A. Londero, et al. 🇧🇷 Animal Reproduction Science 215 (2020) 106309 4.3. Effects on variables during incubation

Fertility was higher among breeder hens fed OMM compared to hens fed BD in the present study. These results are consistent with the findings of Rutz et al. (2003), where it was reported that supplementation with organic minerals (Se-0.2 ppm, Zn- 30 ppm and Mn- 30 ppm) in the diets of broiler breeder hens increased fertility when compared to feeding with inorganic forms (Se0.3 ppm, Zn-100 ppm and Mn-100 ppm). The increase in egg fertility can be attributed to a greater use of minerals involved with fertilization, such as Zn, Mn, Cu and Se. However Yanice et al. (2015) evaluated the effect of supplementation with organic and inorganic mixtures of Mn, Zn, Cu and Cr (chelated to methionine), and reported that there was no difference between groups in terms of fertilized eggs and hatching rates.

Sun et al. (2012) reported that with the supplementation of organic minerals in the diets, there is protection of the breeding hens against lipid peroxidation, greater retention of nutrients in the eggs, and greater growth of the subsequent broiler offspring. In the present study, OMM supplementation in hen diets did not affect embryo mortality and the quality of hatched chicks. Second quality chicks (unhealthy navels, slaughtered and with physical abnormalities) and chick weight (high quality chicks) were not affected by treatments.

The trace minerals Zn, Mn, and Cu play important roles in embryo development, as well as egg hatchability (Kidd et al., 1992), and there is a positive association between Zn content in eggs and egg hatching ability. of hatching eggs. Testosterone metabolism must occur for normal testes growth, sperm production, motility, and sperm count, with relatively less estrogen in the reproductive tissues of male animals (Amen and Al-Daraji, 2011). Supplementation of laying hen diets with organic selenium improves the hatchability of fertilized eggs (Hanafy et al., 2009), and the percentage of fertility and hatchability (Osman et al., 2010). As previously described in this manuscript, there are positive roles of minerals in the reproductive processes of breeding hens, which was confirmed in the present study due to the increase in egg fertility, although there was no effect on the hatchability of these eggs.

5. Conclusions

In conclusion, in the present study, supplementation with Mn, Zn, Fe, Cu and Se can be used for laying breeder hens without affecting the primary variables associated with performance and egg quality. There was a positive effect with the tendency to increase egg production among birds fed organic microminerals throughout the experiment period. Egg fertility was higher with OMM feeding. Among roosters, OMM feeding resulted in increased sperm vigor without changing values for the other semen-associated variables that were evaluated.

Financing

There were no specific grants from public, commercial, or not-for-profit sector funding agencies for the research reported in this manuscript.

Declaration on Conflict of Interests

We, researchers from the Federal University of Santa Maria, declare that there is no conflict of interest in this publication, as we are aware of the seriousness and proper conduct of the Animal Reproduction Science publication.

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