Sunower Seed Meal and Probiotics in Short-term Feeding of Laying Hens

Horticultural byproducts may be used to partially or completely replace more expensive soy and corn while providing adequate energy and protein for broilers and laying hens. Probiotics, such as lactic acid bacteria, may aid in digestion of brous byproducts such as sunower seed meal containing complex carbohydrates that along with some amino acids may not be easily digested by monogastric animals. Thus, byproducts and probiotics, when fed to poultry, may improve the production of nutritious meat and eggs. White Leghorn Crosses (64 layers at 65- to 74-wk-old) were fed one of four diets for four weeks. Diets were (1) a corn/soy Control, (2) Control + 20% sunower seed meal (SFM), (3) Control + Probiotics (Lactobacillus plantarum, rhamnosus, and paracasei - each at > 23.3 Mil CFU/g for a total of approximately 70,000,000 Mil CFU/g added in drinking water), and (4) Control + 20% SFM + Probiotics. Signicance (P < 0.05) and trends (P < 0.10) were determined for production measurements as well as external and internal egg quality parameters. Diet did not signicantly affect production measurements. There were trends due to Probiotics*Week for FCR and SFM*Probiotics*Week for feed intake. For external egg quality, SFM signicantly increased egg weight, decreased specic gravity, and caused a downward trend for egg shell thickness. SFM*Week produced a signicant effect on specic gravity. Probiotics signicantly increased egg weight and egg shell weight while decreasing egg shell thickness; there was a trend due to temporal effects on specic gravity. For internal egg quality, SFM, SFM*Week, Week, and SFM*Probiotics*Week signicantly affected yolk color. Week affected all internal measurements and SFM*Week caused weekly uctuations, thereby producing a trend for Haugh unit. Future in needed to assess production and egg quality parameters when feeding various ber types, the digestibility of SFM/Probiotic diets, and colonization of varying quantities of probiotics (added in water and feed) in the gut of various types and ages of laying hens.


Probiotics in Drinking Water
Sources of (1) water for mixing probiotics and (2) the viable count of bacteria added were analyzed by Michelson Laboratories, Inc. (Commerce, CA, USA). Available water was double distilled, distilled, and chlorinated. The manufacturer (Living Streams Mission, Athol, Idaho) of probiotics also advised using chlorinated water with CuSO 4 (1 ppm) for mixing of bacteria. Heavy metals in chlorinated water were analyzed (UC Davis Analytical Laboratory, Davis, CA). As discussed in Results below, probiotics -active Lactobacillus plantarum, rhamnosus, and paracaseiwere added daily to chlorinated water at > 23. 3 Mil CFU/g, totaling approximately 70,000,000 Mil CFU/g.

Diet
White Leghorn Crosses (64 hens ranging in age from 65-to 74-week-old) were monitored to ensure consistency of weight and egg production.
Data was collected for weight (two weeks) and egg production (nine days) prior to beginning the study. Layers of similar weight (1.59 kg, RMS = 0.0804, P < 0.05) and egg production of (5.67 eggs per week, RMS = 1.391, P < 0.05) were randomly divided into four treatments × four replications × four laying hens/replication and fed either the Control (a corn/soy diet), Control + 20% SFM, Control + Probiotics in drinking water, or Control + 20% SFM + Probiotics in drinking water for four weeks [14,   A power analysis was conducted to determine the adequate number of layers for this study. With a con dence interval at 50% (for which one-half of the layers were fed probiotics) and a 95% con dence level, a population of 64 layers was determined to be adequate (22). Data as a 2 × 2 factorial (Probiotics*SFM, n = 6) in an unbalanced mixed model design with Week as a repeated measure were analyzed using the Proc Mixed Procedure and the PDMIX800SAS Macro [23]. Probiotics (treatment name) was the main effect and SFM (treatment name) became the subplot effect. Pairwise comparisons of treatment means were conducted [23]. The Tukey-Kramer Adjustment was used to assess differences among means at P ≤ 0.05. Signi cance at P ≤ 0.10 was noted. As noted in Table 3, lactic acid bacteria was 270 count in double distilled water (not readily available at the grow-out facility) and 160 count in both distilled water from the laboratory and chlorinated drinking water in lines at the grow-out facility (Method COM ED.4 19.521, Michelson

Probiotics in drinking water
Laboratories, Inc., Commerce, CA, USA). Probiotics from the manufacturer were analyzed and found to contain the quantities noted (~56.7g at 4 billion CFU). For ease of mixing and to avoid changes in the mineral content of diets, Probiotics were mixed in chlorinated drinking water, each at > 23.3 Mil CFU/g, totaling approximately 70,000,000 Mil CFU/g. Analysis revealed no concentration of heavy metals to affect the viability of probiotics in chlorinated drinking water (Table 4).  plantarum, and L. rhamnosus) in chlorinated drinking water.
No mortality occurred during the four week study. SFM, Probiotics, and SFM*Probiotics had no effect on feed intake, egg production, or FCR while Week was signi cant for all production measurements (Table 5). There were trends due to SFM*Probiotics*Week for feed intake (p = 0.0615) and Probiotics*Week for FCR (p = 0.0979) caused by uctuations in measurements over time. SFM signi cantly increased egg weight (p = 0.0242) while decreasing speci c gravity (p = 0.0082). There was a downward trend (p = 0.0581) for eggshell thickness. Temporal uctuations were observed for SFM*Week where speci c gravity was signi cantly affected (p = 0.0301) as shown in Tables 6 and 7. Due to temporal changes, SFM*Probiotics produced a trend for speci c gravity at p = 0.0683. For Probiotics, there was a signi cant increase in egg weight (p = 0.0411) and egg shell weight (p = 0.0441) and a signi cant decrease in egg shell thickness (p = 0.0109). Speci c gravity trended downward at p = 0.0789. Week was signi cant for egg weight (p = 0.0010), egg shell weight (p = 0.0011), and egg shell thickness (p = 0.0280); it was highly signi cant for speci c gravity (p = < 0.0001) and there was a temporal effect for shape index (p = 0.054). Probiotics*Week and SFM*Probiotics*Week had no effect on external egg quality parameters (Table 6).   10.3%) on laying hens for weight gain, feed consumption, egg production, and FCR [1]. Our ndings and those of other investigators suggest that while SFM did not improve production parameters, there were no detrimental effects. Thus, SFM could be substituted for soybean meal in the diets of older layers.

Production: Probiotics
In our work, there were no effects of Probiotics alone on production parameters while results from other investigators reported improvements.
Investigators reported that the average time for colonization of the gut by various B. Subtilis species was three to six weeks in older hens [30]. Our results for Lactobacillus species seem to indicate that substantial colonization in the gut of older layers needs a period longer than 4 weeks to affect production measurements. Possibly, on-going competition of the Lactobacillus species with established organisms may have caused uctuations in weekly results as discussed below for external and internal egg quality measurements. Alternatively, the quantity of bacterial species used may not have produced a signi cant enhancement for production measurements even after colonization in hens fed in our study.
As noted above, a lysine de ciency, a low digestion coe cient for lysine, and a negative correlation between ber content and total metabolizable energy of SFM were reported [4,7]. Probiotics (including the Lactobaccilus species) as live microbial non-digestible supplements can colonize in the intestine of animals as well as in the ceca of poultry [8]. If colonized, they increase amylase, causing catalysis of starch to sugars [11]. Also, due to enhanced digestibility and absorption of nutrients (including amino acids), they can improve feed e ciency, productivity of laying hens, and egg quality measurements [12]. Due to our hypothesis that Probiotics would improve digestion of SFM brous material and amino acids, there was no attempt to adjust digestible lysine and other amino acids across diets. No effect for SFM*Probiotics indicated that Lactobacillus species did not greatly enhance production parameters by increasing digestion of lignocellulosic compounds or digestibility of amino acids in SFM. Future work will include results for apparent digestibility of all diets to more accurately measure improvements in amino acid availability caused by Lactobacillus species alone and when coupled with SFM.
It was assumed that uctuations by Week for feed intake and FCR of older layers likely contributed to trends noted for SFM*Probiotic*Week.  [26]. Yalcın et al. (2008) revealed that egg weight of hens fed a SFM or a soybean meal diet with 0 and 2 g/kg commercial yeast culture product (Saccharomyces cerevisiae) for 16 weeks did not change [25]. Laudadio et al., 2014 noted that egg weight was statistically similar among eggs of hens fed soybean meal or a low-ber SFM diet (CP, 42.3%; CF, 10.3% and IR, 16%) for 10 week [1]. These contradictory ndings suggest that effect of ber types and quantities on external egg quality should be thoroughly investigated.
In our study, the decrease in speci c gravity caused by SFM and SFM*Week was likely associated with the trend toward a decrease in egg shell thickness and greater egg mass due to age and less calcium deposition [32,33]. While eggshell thickness for SFM trended downward in the present study, it did not fall below 0.33 mm, indicating that eggs had shells of adequate thickness that could withstand the rigors of marketing [34].
SFM had no effect on shape index. A high shape index indicates more eggs of rounded shape as opposed to more elongated ones [35]. Age by strain and season may have an effect on index with those from older hens and laid in summer and autumn having an elongated shape [35,36]. However, all eggs in our study had an index of 84. Eggs with an index > 76 can withstand the rigors of processing/packaging and transportation [35,36].

External egg quality: Probiotics
Our nding for an increase in egg weight produced by Probiotics did not agree with those of Kurtoglu et al. (2004) who noted no effect [28]. However, Mohan et al. (1995) noted an improvement in egg weight for hens fed diets supplemented with probiotics for 10 weeks during the peak laying period [27].
As Probiotics increased egg weight, eggshell weight increased and eggshell thickness decreased. The decrease in eggshell thickness was validated by the trend toward a decline in speci c gravity [33]. SFM*Probiotics affected speci c gravity and eggshell weight; also, this may have been in uenced by the age of hens. Investigators suggested that probiotics and SFM promoted growth of intestinal micro ora, thereby producing a healthy gut lining causing improved digestion and subsequent deposition of nutrients in yolk (and greater weight) although mechanisms are not understood [37,38]. More work with younger birds and/or greater quantities of Probiotics may help clarify these ndings. Fat at two times more in the SFM diet than in the Control may have caused some dilution of color as well.
The signi cant effect of SFM*Week on the yolk:albumen and proportional albumen may have indicated greater internal weight of the egg associated with increasing age. Even though there was greater mass over time, the Haugh unit, the standard for overall internal egg quality, remained low throughout the study. A Haugh unit for young layers is > 91; that for older layers in our work was ~ 73. Fluctuation in Haugh units over time, as with some production and egg quality measurements, may have been related to intermittent colonization of Lactobacillus species during the 4-week period.

Conclusion
For signi cance at P < 0.05, neither SFM, Probiotics, nor SFM*Probiotics affected production measurements. SFM increased egg weight and decreased speci c gravity. The increase in egg and shell weight, was accompanied by a decrease in eggshell thickness, likely compounded by less calcium deposition as hens aged. A decrease in yolk color was due to addition of SFM that reduced pigments; increased fat content in SFM diets likely caused a diluting effect as well. SFM*Probiotics affected some external egg quality measurements at P < 0.01, but had no effect on internal egg quality. Week (age of hens) affected production and external/internal measurements. More work is needed to clearly establish (1) the effect of various ber types on production and egg quality parameters, (2) the digestibility of SFM/Probiotic diets, and (3)