Evaluation of industry strategies to supply dietary chelated trace minerals (Zn, Mn, and Cu) and their impact on broiler breeder hen reproductive performance, egg quality, and early offspring performance

Rearing

Laying Period

At wk 21, pullets (n = 1,290 pullets) were assigned to a laying pen (n = 30 pens; n = 43 birds per pen; 2.4 × 3.6 m²) with each pen having similar mean BW and uniformity along with 3 Cobb Vantage males in each pen. To increase this flock’s mating frequency, one additional male was included in each pen at 32 wk of age. In addition (n = 151), Cobb Vantage spike males were grown to 21 wk of age, and methodically used to replace old males in pens across all treatments starting at 54 wk of age through the end of the experiment. Two-thirds of the floor space was covered by slats, and the remaining third of the pen was covered with pine shavings. A 6-hole nest section was placed on the slats of each pen. Hens were fed using Chore-time (cat no. CT-2565/201503, Chore-time Inc., Milford, IN) pan feeders (n = 4 per pen) with an exclusion opening to prevent males from accessing the hen feeder. Pan feeders were filled with feed everyday according to daily allowance and hand lowered every morning at 6:30 am. Males were fed from plastic (4 hole) suspended feeders (cat no. MF-2BX Fortex-Fortiflex, PR) over the shavings area. All birds had free access to water from a nipple drinker line. A sample of hens and roosters was individually hand-weighed weekly (n = 3 pens per treatment) to adjust feed allowance based on BW gain, and in the case of hens, egg production increase or decrease. Weekly average hen BW, and BW uniformity were calculated. Hens in all 30 pens were fed the same amount across treatments throughout the experiment (80–157 g per bird). Likewise, roosters were fed the same amount through the end of the experiment (91–139 g per male).

Laying Dietary Treatments

Egg Production and Egg Weight

Eggs within a pen were collected 4 to 5 times a day, graded and recorded. The egg production per pen was expressed as weekly hen-day egg production (%). A flat of settable eggs per pen was weighed weekly to determine average egg weight per pen.

Eggshell Quality

Shell quality was evaluated by the specific gravity floatation method (n = 1 days-worth of settable eggs per pen; n = 10 pens per treatment) during wk 30, 34, 38, 42, 46, 50, 54, 58, 62, and 64. Fresh eggs (laid the day before) collected for this procedure were stored in the same room as the salinity tanks for approximately 15 h to allow the eggs to come to the same temperature as the solutions. Eggs sorted by pen were floated in water tanks with different salinity levels ranging from 1.060 to 1.095 g/cm³ (increases of 0.005 g/cm³). Thin and thick shell eggs floated in low and high salinity tanks, respectively. A weighed mean shell quality score was determined by pen with the formula described below, where the number of eggs that floated in each tank (E_i) were multiplied by the tank’s salinity concentration (S_i). The products of each tank’s multiplication (E_i × S_i) were be added and divided by the total number of eggs per pen used (Et). This analysis was performed on 2 separate days after wk 50 to increase the sample size evaluated as egg production declined.

Eggshell Cuticle

Eggshell cuticle quantification was evaluated as described by previous researchers (Bain et al., 2013, 2019) with minor modifications, during the same week eggshell quality was evaluated. Ten eggs per pen were collected and stored at room temperature (21°C) overnight. Three equator regions of the eggshell were measured with a Minolta Spectrophotometer (model CM 700-D, Konica Minolta Inc., Tokyo, Japan) to determine unstained eggshell cuticle reflectance (Ref_pre; %) at 650 nm. Reflectance % is the ratio of light leaving the spectrometer that comes back to the device after striking the eggshell surface. The eggs were covered in blue MST cuticle dye solution for 15 min (M.S. Technologies Ltd., Northamptonshire, UK) and rinsed in buckets with tap water 3 times to remove excess dye (3 min each). Eggs were left to dry overnight on plastic flats and poststain cuticle reflectance (Ref_post) was determined on same 3 equator regions of the egg. When subtracting Ref_pre and Ref_post, the cuticle deposition value obtained (+∆Ref) represents how much light is being captured on the stained eggshell cuticle and usually ranges between 20 and 30% in commercial layer and broiler breeder eggs (Bain et al., 2013, 2019). Therefore, a higher ∆Ref indicates more stained cuticle presence on the eggshell. The triplicate values of ∆Ref were averaged for each egg.

Egg Minerals

Egg contents (n = 1 egg per pen; yolk + albumen) and yolk (n = 4 yolks composite per pen) samples were collected from hen pens at 30, 40, 50, and 60 wk of age, stored in individual containers, hand-mixed, and shipped in a cooler with ice packs. Egg contents and yolk composites were analyzed by ATC Scientific (North Little Rock, AR) for Zn, Mn, and Cu mineral contents as described by AOAC 985.01 procedures and expressed as ppm (as-is basis; AOAC International, 2005c).

Incubation

Settable eggs (n = 90 per pen) were incubated 10 times by pen during the laying period every 4 wk, starting at wk 29 through wk 65. Eggs were set in Natureform incubators (cat. no. NMC-2000, Natureform Inc., Jacksonville, FL) at 37.5°C (53% of relative humidity) and rotated 45° every 2 h during the first 18 d of the 21 d of incubation period. To determine fertility and early embryo mortality, eggs were candled 11 d after being set, and transferred to a hatcher at incubation d 18 with temperature set to 37°C for the remaining 3 d. Hatchability and embryo analysis was performed at every hatch, and in addition chicks were weighed at 4 different hatches (33, 41, 57, 65 wk).

Broiler Grow-Out and Tibias

Hatches at 33, 41, 57, and 65 wk were (n = 30 broiler chicks per pen) placed in floor pens covered with pine shavings and fed a common broiler starter diet for 7 d (pelleted and crumbled; 21% CP and 2.97 Mcal per kg). Chicks were placed in pens in correlation to each maternal pen (n = 30 broiler pens). Temperature was reduced from 32°C to 27°C through d 7 according to bird comfort in a light-tight broiler house. During the first day, 24 h of light were kept, then reduced to 23 h per d through d 7 (27 Lux). Chicks had access to water using a nipple drinker line. Chick’s growth performance was determined by weighing whole pens (n = 30 chicks per maternal pen) at placement and after 7 d of grow-out, and by determining residual feed at d 7. Mortalities were recorded daily to calculate the cumulative mortality. Additionally, left tibias (n = 6 per pen) were collected from grown chicks from the 41-wk hatch, shipped on ice, and analyzed by ATC Scientific (North Little Rock, AR) for Zn, Mn, and Cu using same AOAC 985.01 method used with eggs. Tibia ash content and Ca were determined as well using AOAC 942.05 and AOAC 2011.14 MOD methods, respectively (AOAC International, 2005a,b).

Bone Mineral Density

Bone mineral density of chicks and hens was assessed with a dual energy X-ray absorptiometry (DEXA) scan using a Lunar Prodigy scanner (cat. no DF15942, GE Healthcare, IL) as described in previous experiments (Schallier et al., 2019). Chicks (n = 10 per maternal treatment) were euthanized via CO₂ asphyxiation after 3 separate hatches (33, 41, and 57 wk) and after the 7 d grow-outs for 2 hatches (41 and 57 wk) before being scanned. A similar procedure was performed to scan hens at ages of 30, 45, and 60 wk of age (n = 10 per treatment each timepoint), with the only difference being that hens were first stunned via electrocution, and then euthanized using cervical dislocation.

Statistical Analysis

Data were analyzed using a generalized linear model (GLM) for analysis of variance and a least mean square (LSMEANS) for mean differences evaluation using SAS version 9.4 (SAS Institute, Cary, NC). Means were declared different when P ≤ 0.05. Both breeder age and the interaction between diet × breeder age were also analyzed as main effects, and their P value will be shown in the figure captions and table footnotes.

Breeder Hen Performance

Percentage egg production of hens consuming the treatment diets can be seen in Figure 1. Hens reached 5% egg production at the end of wk 25 and were provided the treatment diets at the beginning of wk 26. On average, hens peaked in egg production (81%) at 31 wk of age (age effect P < 0.001). Egg production was greater during wk 39, 64, and 65 in hens fed the MIX diet (P ≤ 0.029) compared to hens fed the ORG diet, and both MIX and CTL hens had higher egg production compared to ORG hens at wk 48, 56, and 57 (P ≤ 0.037). Additionally, results indicated that the laying rate of those hens fed CTL diet was more similar to the MIX hen performance than that of the ORG hens. Table 3 shows the cumulative broiler breeder hen performance metrics obtained through the laying period, and reiterates that overall egg production percentage was higher in MIX hens (56.1%), followed by CTL hens (55.3%), and then the ORG hens (53.6%; P < 0.001).

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Figure 1. Effect of broiler breeder hen dietary trace mineral inclusion on weekly hen-day egg production (%) from wk 25 to 65. The 3 laying treatment diets provided Zn, Mn, and Cu (mg/kg): CTL = predominantly inorganic trace minerals (143–180–123); ORG = organic HMTBa trace minerals (ORG: 50–60–15); and MIX = a blend of inorganic/organic HMTBa trace minerals (75/25–90/30–22.5/7.5). These diets were fed starting at wk 26 until the end of the experiment. Each dietary treatment was represented by n = 10 pens with 43 initial hens per pen. Egg production was higher during wk 39, 64, and 65 in hens fed the MIX diet (P ≤ 0.029) compared to ORG hens only, and both MIX and CTL hens had higher egg production compared to ORG hens at wk 48, 56, and 57 (P ≤ 0.037). Significant differences between treatments within age (P ≤ 0.05) indicated by (*). Age effect P < 0.001; age × diet effect P = 0.967.

Table 3. Effect of dietary trace mineral supplementation on overall breeder hen cumulative performance through wk 65.

Variable	Dietary treatment2			SEM	P value
	CTL	ORG	MIX
BW, g	3,938b	3,990a	3,946b	11.3	0.002
CV, %	11.5ab	11.7a	11.1b	0.1	0.019
Mortality, %	6.1	6.3	6.3	0.1	0.989
Egg production, %	55.3b	53.6c	56.1a	1.6	<0.001
Eggs per hen housed	162	156	166	3.6	0.208
Feed conversion (kg:doz. eggs)	3.169	3.284	3.146	0.1	0.354
Egg weight, g	63.0b	63.7ab	64.0a	0.3	0.038
Fertility, %	83.3	84.9	83.3	1.6	0.726
Hatch of fertile, %	89.4	90.0	90.5	0.6	0.444
Hatchability, %	75.6	76.7	76.1	1.7	0.898
Chicks per hen housed1	121	119	126	4.8	0.576
Hatched chick weight, g	45.1	45.2	45.5	0.5	0.832

1

Calculated using weekly cumulative egg production and hatchability ≥wk 27.

2

The 3 laying treatment diets provided Zn, Mn, and Cu (mg/kg): CTL = predominantly inorganic trace minerals (143–180–123); ORG = organic HMTBa trace minerals (50–60–15); and MIX = a blend of inorganic/organic HMTBa trace minerals (75/25–90/30–22.5/7.5). These diets were fed starting at wk 26 until the end of the experiment. Each dietary treatment was represented by n = 10 pens with 43 initial hens per pen.

a–c

Means with different superscripts within rows denote significant differences (P ≤ 0.05).

Hens fed the ORG diet were 1.3 and 1.1% heavier compared to those fed CTL and MIX, respectively (P = 0.002; Table 3), even though pen placement mean BW was similar at 21 wk of age. Since all hens were fed equal feed amounts, this indicates that ORG birds laid fewer eggs but in turn used more nutrients toward BW gain. Improved BW uniformity was detected in the MIX group compared to ORG hens (11.1 vs. 11.7%; P = 0.019). In addition, eggs from MIX-fed hens were heavier compared to CTL hens (64.0 vs. 63.0 g; P = 0.038), although were of similar weight compared to eggs laid by ORG hens.

One probable reason why the egg production of CTL-fed hens was similar to those fed MIX is because generally, both diets provided similar or high Zn, Mn, and Cu levels when compared to this strain recommendations (Cobb-Vantress, 2020). The improvement in egg production by combining trace mineral sources (MIX) agrees with those reported by Hudson et al. (2004), where breeders fed a mixture of Zn from both inorganic (Zn sulfate: ZnSO₄) and organic sources (Zn amino acid complex) had improved egg production compared to those fed ZnSO₄ only (57.3 vs. 56.7% egg production; P < 0.05). In contrast, feeding a breeder diet containing reduced levels of organic amino acid-complex trace minerals did not affect reproduction in Araújo et al. (2019) study. Although, Araújo et al. (2019) reported that the organic trace mineral diet provided: 55% of Zn, 58% of Mn, and 72% of Cu compared to their inorganic diet, which were substantially higher than those provided in our experiment by the ORG diet: 35% of Zn, 33% of Mn, and 12% of Cu compared to our CTL diet.

Explaining the role of our dietary treatments on hen performance is difficult as these were industry levels provided by different sources. Sufficient dietary Zn is necessary for high egg-laying rate since is a crucial component of steroid hormone receptors and assists in maintaining reproductive hormones in breeder hens (Schwabe and Rhodes, 1991; Prabakar et al., 2021). It is possible that the reduced Zn provided by ORG diet (50 mg/kg) was not enough to outperform the high industry levels supplied with the CTL (143 mg/kg) and MIX diets (100 mg/kg), which are closer to Cobb-Vantress (2020) recommended levels of 110 mg/kg. For Mn levels, a dose-response study demonstrated that the inorganic Mn (as sulfate) requirement for optimal performance in laying breeders is 97 mg/kg but providing 128 mg/kg can aid in improving eggshell quality (Noetzold et al., 2020), being similar to Cobb-Vantress (2020) recommendation of 120 mg/kg. The CTL diet Mn levels (180 mg/kg) exceed these requirements, although the Mn levels in the MIX diet (120 mg/kg) are closer. Additionally, the CTL diet provided high Cu (123 mg/kg), exceeding this strain recommendation of 10 to 15 mg/kg (Cobb-Vantress, 2020). In broiler nutrition, high levels of TBBC provided at “pharmacological” levels (e.g., 188 mg of Cu per kg) can produce a similar growth performance compared to broilers fed diets containing antibiotic growth promoters (Arias and Koutsos, 2006). However, Cu doses of 65 to 300 mg/kg provided by TBCC in commercial layer hen diets does not impact egg production and overall performance (Liu et al., 2005; Kim et al., 2016), making it unclear whether Cu influenced breeder performance in our study. With information from previous research, it is possible that the higher levels of Zn and Mn provided by our CTL and MIX diet stimulated reproduction when compared to ORG diet.

Eggshell Quality and Cuticle Deposition

Eggshell quality results are shown in Figure 2. Specific gravity of an egg is an indirect indicator of eggshell integrity (Butcher and Miles, 1991), which is crucial for successful egg collection and incubation (McDaniel et al., 1981; Roque and Soares, 1994). Dietary trace minerals are necessary for proper eggshell membrane formation and the supply of carbonate to the eggshell (Chowdhury, 1990; Nys et al., 1999; Xiao et al., 2014). Differences between treatment groups were more apparent as eggshell quality declined with age (age effect P < 0.001). Eggs from hens fed the MIX diet maintained a higher eggshell quality during wk 54 and 58 compared to those fed CTL, but not different from those of ORG hens (P ≤ 0.037). Similarly, Hudson et al. (2004) reported an improvement in shell quality when using solely organic trace minerals or partially replacing ZnSO₄ (mixed sources). It is possible that increasing the specific gravity of eggs laid by MIX hens helped to reduce shell breakage and therefore egg loss during this late period, which contributed to a higher egg production.

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Figure 2. Effect of broiler breeder hen trace mineral dietary inclusion on eggshell quality as determined using specific gravity (g/cm³) method every 4 wk starting at wk 30. The 3 laying treatment diets provided Zn, Mn, and Cu (mg/kg): CTL = predominantly inorganic trace minerals (143–180–123); ORG = organic HMTBa trace minerals (ORG: 50–60–15); and MIX = a blend of inorganic/organic HMTBa trace minerals (75/25–90/30–22.5/7.5). These diets were fed starting at wk 26 until the end of the experiment. Each dietary treatment was represented by n = 10 pens of 43 initial hens each. Eggs from MIX-fed hens had an improved shell quality during wk 54 and 58 compared to those from hens fed CTL (P ≤ 0.037). ^a,bMeans within each given timepoint (wk of age) with unlike letters are declared statistically different (P ≤ 0.05). Error bars represent ± SE by within age. Age effect P < 0.001; age × diet effect P = 0.088.

Eggshell cuticle is mainly composed of glycoproteins and coats the shell 1.5 to 2 h prior to oviposition, offering physical and antimicrobial properties to eggs during storage and incubation (Breen and De Bruyn, 1969; Board and Halls, 1973; Wedral et al., 1974). Cuticle deposition is a heritable trait (Bain et al., 2013) and is unclear if nutrition can affect it. Our results displayed in Figure 3 indicate that there were minimal changes until eggshell cuticle reaches a maximum value at wk 50 as affected by age (P < 0.001). We only detected differences at wk 50, where hens consuming the MIX diet had increased eggshell cuticle deposition compared to those from hens fed CTL or ORG diets (P = 0.019). Given the role of estrogen receptors on oviduct maintenance (Munro and Kosin, 1943), and their association with cuticle deposition (Bain et al., 2013), it is probable that sufficient dietary trace mineral has a stimulatory impact on eggshell quality but also cuticle deposition.

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Figure 3. Effect of broiler breeder hen trace mineral dietary inclusion on eggshell cuticle deposition (reflectance difference, %). The 3 laying treatment diets provided Zn, Mn, and Cu (mg/kg): CTL = predominantly inorganic trace minerals (143–180–123); ORG = organic HMTBa trace minerals (ORG: 50–60–15); and MIX = a blend of inorganic/organic HMTBa trace minerals (75/25–90/30–22.5/7.5). These diets were fed starting at wk 26 until the end of the experiment. At each timepoint n = 30 eggs per pen were sampled for a total of n = 100 eggs per dietary treatment. Eggs from MIX-fed hens had an improved cuticle deposition at 50 wk (P = 0.019). ^a,bMeans within each given timepoint (wk of age) with unlike letters are declared statistically different (P ≤ 0.05). Error bars represent ± SE by timepoint. Age effect P < 0.001; age × diet effect P = 0.047.

Mineral Contents of Eggs

Fertility and Incubation

Figure 4 displays the effect of dietary trace mineral supplementation on fertility (Figure 4A) and incubation parameters (Figure 4B: hatchability; Figure 4C: hatch of fertile; Figure 4D: early dead). The fertility and hatchability in natural mating conditions is primarily influenced by the male age, libido, mating, and sperm transfer (Duncan et al., 1990; McGary et al., 2003). Dietary trace mineral supplementation did not affect fertility or hatchability % (P ≥ 0.205). Lower hatch of fertile % was detected at wk 41 when incubating eggs from ORG hens compared to those from CTL and MIX hens (P = 0.018), although this effect was not consistent throughout the rest of the experiment (P ≥ 0.060). In this study, the average peak in fertility was 95% (33 wk; Figure 4A) for all treatments, which is acceptable in commercial settings. However, a dramatic decline in fertility occurred after 41 wk of age until the end of the study (94–59%; Figure 4A), which is not usual in our research unit and commercial U.S. breeder houses per personal communication with field experts, and as it is indicated in guidelines (Cobb-Vantress, 2020). We can confirm from our behavioral observations and industry reports (Graber, 2021a,b), that the poor mating frequency of this particular breeder male strain interfered with the possible influence that dietary trace minerals could have on fertility.

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Figure 4. Effect of dietary trace minerals on breeder egg fertility (A), hatchability (B), hatch of fertile (C), and early dead (D) during incubation. Data from n = 10 separate hatches. Eggs (n = 90 per pen) were incubated and hatched by pen at each given timepoint. The 3 laying treatment diets provided Zn, Mn, and Cu (mg/kg): CTL = predominantly inorganic trace minerals (143–180–123); ORG = organic HMTBa trace minerals (ORG: 50–60–15); and MIX = a blend of inorganic/organic HMTBa trace minerals (75/25–90/30–22.5/7.5). Dietary trace mineral supplementation did not affect fertility or hatchability % (P ≥ 0.205). Lower hatch of fertile % was detected at wk 41 when incubating eggs from ORG hens compared to those from CTL and MIX hens (P = 0.018), although this metric remained unaffected throughout the rest of the experiment (P ≥ 0.060). Control (CTL) and MIX diets reduced early embryo mortality at wk 41 (P = 0.004), but in contrast, both MIX and ORG diets reduced this number at wk 65 (P = 0.024) compared to feeding CTL to hens. Mid and late embryonic mortalities remained unaffected through the experiment (P > 0.05; data not shown). ^a,bMeans within each given timepoint (wk of age) with unlike letters are declared statistically different (P ≤ 0.05). (A) Age effect on fertility P < 0.001; Age × diet P = 0.731. (B) Age effect on hatchability P < 0.001; age × diet P = 0.590. (C) Age effect on hatch of fertile P < 0.001; age × diet P = 0.045. (D) Age effect on early dead P = 0.691; age × diet P = 0.047. Error bars represent ± SE by timepoint.

Trace mineral carryover from hen to egg is necessary for proper embryo trace mineral mobilization, and development (Richards, 1997; Favero et al., 2013). We observed a higher number of early embryonic deaths in eggs from ORG hens collected at wk 41 (P = 0.004; Figure 4D) when compared to the eggs from CTL and MIX hens, although these results were not consistent during the remainder of the study. Lastly, early embryonic mortality was reduced in eggs collected at wk 65 from ORG and MIX hens (P = 0.024; Figure 4D) compared to eggs from CTL hens.

Broiler Offspring Performance and Tibias

Hen and Chick Bone Mineral Density

The results of bone mineral densities obtained with DEXA scans of hens, hatched chicks, and grown chicks are shown on Figure 5A to C, respectively. Hens in the 3 dietary groups had a similar bone mineral density at all ages sampled (P ≥ 0.089). Even with no differences in chick performance, our scan results showed that hatched chick’s bone mineral density was improved when obtained from ORG-fed hens when fertile eggs were collected from young hens (29 wk; P = 0.004; Figure 5B). Bone density was higher on 7-day grown broilers from the wk 41 hatch when obtained from ORG hens compared to those from MIX-fed hens (0.076 vs. 0.072 g/cm²; P = 0.047; Figure 5C) but were not different from those of CTL-fed hens. Our results concur with previous studies, where breeders supplemented with organic trace minerals (DL-methionine or HMTBa, respectively) positively influenced offspring tibia and femur bone integrity (Kidd et al., 1992; Torres, 2013).

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Figure 5. Effect of dietary trace mineral supplementation on breeder hen (A), hatched broiler (B), and 7-day-old broiler total bone mineral density obtained using DEXA scans. The 3 laying treatment diets provided Zn, Mn, and Cu (mg/kg): CTL = predominantly inorganic trace minerals (143–180–123); ORG = organic HMTBa trace minerals (ORG: 50–60–15); and MIX = a blend of inorganic/organic HMTBa trace minerals (75/25–90/30–22.5/7.5). At wk 30, 45, and 60, n = 10 hens per treatment were sampled. Chicks (n = 10 per maternal treatment) hens of 29, 41, and 57 of age were sampled at hatch, and another 10 chicks per maternal treatment after being grown for 7 d when obtained from hens of 41 and 57 wk of age only. Hens from the 3 dietary groups had a similar bone mineral density at all ages sampled (P ≥ 0.089; A). Hatched chick’s bone mineral density was higher when obtained from 29-wk-old ORG compared to CTL and MIX hens of the same age (P = 0.004; B). Bone density was improved on 7-day grown broilers from the wk 41 hatch when obtained from ORG hens compared to those from MIX-fed hens only (0.076 vs. 0.072 g/cm²; P = 0.047; C). ^a,bMeans within each given timepoint (wk of age) with unlike letters are declared statistically different (P ≤ 0.05). Error bars represent ± SE by timepoint. (A) Hen bone mineral density age effect P < 0.001; Age × diet P = 0.408. (B) Hatched broiler bone mineral density hen age effect P = 0.041; age × diet P = 0.405. (C) Seven-day-old broiler bone mineral density hen age effect P = 0.442; age × diet P = 0.278.

The enhancement of performance and eggshell quality obtained when combining organic and inorganic trace minerals in the breeder diets like in our MIX treatment agrees with earlier reports (Hudson et al., 2004; Favero et al., 2013), although the explanation of this phenomenon is still unknown. The chemistry of HMTBa trace minerals may facilitate their stability and uptake in the gut lumen (Leeson and Summers, 2005). However, trace mineral bioavailability is studied using markers that may not be crucial in breeders (Forbes and Erdman Jr, 1983; Brown and Zeringue, 1994; Cao et al., 2000), and perhaps is also influenced by breeder age. Organic and inorganic trace minerals are absorbed from the lumen to bloodstream via 2 different routes, respectively (Goff, 2015, 2018): 1) transcellular mechanism, or 2) paracellular mechanism. Whether absorbing these minerals via both routes is advantageous to breeder reproduction is unclear but is a potential explanation of our results.

In large broiler experiments (Manangi et al., 2012), replacing the diet inorganic levels of 100, 90, 125 mg/kg of Zn, Mn, and Cu, respectively, with 32, 32, and 8 mg/kg of HMTBa trace minerals was effective in maintaining similar broiler performance and reducing mineral excretion in the litter. In our trial, hens fed reduced doses of HMTBa (ORG), had offspring with higher bone mineral density, although the egg yolk Mn carryover, and overall performance data results suggest that these levels were not sufficient to outperform those high industry levels provided in the CTL and MIX diets. It is not possible to distinguish whether dose or source of trace minerals used induced the responses shown in our study. To address these questions, future dose-response experiments should compare equal mineral levels among treatments provided by different sources (organic or inorganic). As shown in previous literature, reducing and replacing inorganic trace minerals in breeder diets are a potential tool to maintain or enhance reproduction and offspring quality (Torres, 2013; Araújo et al., 2019), although it should be done carefully when replacing high industry safety levels.