Reproductive and Physiological Responses and Egg Quality Traits of Isa Brown Chickens fed Diets Supplemented with Ginger or Turmeric Powder

The increasing demand for healthy and low fat poultry products by consumers has necessitated the use of natural growth promoters to enhance hens’ laying performance. This study investigated the reproductive response and egg quality traits of pullet chickens fed dietary turmeric (Curcuma longa) and ginger (Zingiber ocinale) powder. Five hundred and four pullets were assigned basal diet (G 0 T 0 ) or basal diet supplemented with 1.5 (G1.5) and 3% (G3) ginger or 1.5(T1.5) and 3% (T3) turmeric to investigate the reproductive responses and egg quality traits of pullet chickens during a 60 week trial period. Data were collected on egg and laid out in a Completely Randomized Design in a 2 x 3 factorial arrangement. Hen-day and hen-housed egg production, egg mass, feed conversion ratio and age at rst lay were signicantly (p<0.05) affected by phytobiotic type. The best (p<0.05) hen-day, hen-housed, egg mass, FCR, earliest age at rst lay and least weight at rst lay were obtained in birds placed on 1.5% ginger ration. Percentage mortality was not signicant except at the different inclusion levels and was least among birds fed 1.5% phytobiotic inclusion levels. The highest (p<0.05) value for egg length, egg width, egg shape index, albumen weight and yolk colour were obtained in birds fed 3% turmeric diets. Internal egg qualities were most enhanced by turmeric inclusion while dietary ginger improved the external qualities. The study concluded that dietary ginger and turmeric enhanced reproductive performance and egg quality of egg-type chickens fed over their commercial production lifespan without impairing their overall well-being. hen-housed FCR. Hen-day egg (p<0.05) compared to for hens fed diets and 1.5% turmeric Hen-housed placed on ginger diets at inclusion which 62.26%, not statistically from fed turmeric


Introduction
The ever-increasing global population and the increase in individual food consumption have continuously made the demand for animal products to be on the rise. The world's per capita consumption of eggs has now attained almost a double-fold of what obtained in the early 1960s, while poultry meat consumption has increased by 50% (FAO, 2020). including gingerone and shogaol of ginger (Fuhrman et al., 2000) and curcuminoid compounds of turmeric plants (Chattopadhyay et al., 2004), having ability to lower egg yolk lipids. There has been a growing concern on the use of antibiotics as growth promoters due to the residues in animal tissues and the production of drug resistant bacteria (Zomrawi et al., 2013) that are zoonotic in nature. Hence, the use of phytobiotics is now increasingly gaining a global approval especially in the aspects of health and nutrition as a possible suitable alternative.
Ginger and turmeric powder may improve nutritive value of livestock diets, enhance animal performance by increasing their growth rate, better feed conversion e ciency, improved livability and lowered mortality in poultry birds (Devegowda, 1996).
Haematological studies are of ecological and physiological importance which aids the understanding of the relationship of blood characteristics to the environment (Ovuru and Ekweozor, 2004). It could also be useful in the animal selection, especially those that are genetically resistant to certain diseases and environmental conditions (Mmereole, 2008;Isaac et al., 2013). Haematological parameters are good indicators of the physiological status of animals and valuable in monitoring feed toxicity especially with feed constituents that affect the blood as well as the Loading [MathJax]/jax/output/CommonHTML/jax.js health status of farm animals (Oyawoye and Ogunkunle, 2004). The safety of turmeric and its characteristic yellow colouration are endorsed by organizations and researchers (WHO, 1987;Hallagan et al., 1995;NCCAM, 2012). The active ingredient (curcumin) has hepatoprotective properties (Pal et al., 2001) and is claimed to enhance digestion and metabolism of nutrients. Ginger rhizome also contains a number of compounds that exert varying biological activities, including antioxidant, antimicrobial (Akoachere et al., 2002) and various pharmacological effects (Ali et al., 2008).
Generally speaking, herbs could serve as feed additives due to their suitability and preference, reduced risks of toxicity and minimum health problems (Devegowda, 1996). Several studies have been carried out to verify the e cacy of these feed additives (Mishra and Singh, 2000;Deepak et al., 2002;Jahan et al., 2008). There is however con icting results in the literature. Most studies have focused on a short term administration of the additives, mostly in broilers and layer chickens alike. Therefore, there is a need to further explore the potency and the impact of their extended use over the reproductive lifespan of egg-type chickens.

Processing of test ingredients
Ginger and turmeric rhizomes were obtained from a reputable local market in Lagos, Nigeria. Ginger rhizomes were washed and cleaned. The cleaned rhizomes were cut into slices and to air-dried until certain moisture content is reached; after which they were pulverised and stored away in an airtight container before being incorporated into the experimental diets. Turmeric rhizomes were also processed as described for ginger. Table 1 shows the composition (%) of the experimental layer diets. Five hundred and four day old pullet chicks were used for the study. The pullets were divided into two equal groups of two hundred and fty two birds each. The rst group of the birds was fed diets containing ginger powder (G) while the other was allocated to diets containing turmeric powder (T). The birds in each group were further divided into three treatment groups of four replicates per treatment with twenty one birds per replicate. The birds fed basal diets (G 0 and T 0 ) without ginger and turmeric powder served as the control groups. Pullets in experimental groups G 1.5 and T 1.5 were fed diets containing ginger and turmeric powder at 1.5% inclusion levels respectively, while those of G 3 and T 3 were fed diets containing ginger and turmeric powder respectively, at 3% inclusion levels.

Experimental diets
Layer mash containing ginger (G) and turmeric (T) powder supplemented at 0%, 1.5% and 3% levels were introduced to laying birds at 5% in lay. The diets were fed to the birds for a period of 37 weeks. The diets were formulated to meet the nutrient requirements of laying birds (NRC, 1994).

Data collection
Egg production parameters Eggs laid per replicate were collected daily and the weights were determined using a Mettler top-loading weighing scale. Body weights of the birds at rst lay were taken as the average weight of live birds over the number of birds per group while age at rst lay was the number of days from day-old to the day the rst egg was laid. The body weights at rst lay and the weight of rst egg were determined using a balance scale with sensitivity of 0.01g. Egg number per week was determined by the total number of eggs laid by individual pullet in each group per week.
Hen-day egg production: Egg production was calculated on hen-day basis per group as:

Numberofeggslaid Numberoflivebirds x100
Hen-housed egg production: Egg production was determined on hen-housed basis per group as:

Numberofeggslaidduringthelayingperiod Totalnumberofbirdshousedatthebeginningoflayingperiod x100
Egg mass: This was calculated per group as:

Totalfeedintake(g) Eggmass(g)
Mortality: This was recorded as they occurred as: External quality of eggs Individual egg was weighed using top-loading weighing balance and their shape indices were measured as egg width (mm) divided by egg length (mm). Egg shell was weighed using a sensitive scale and shell thickness was measured by digital caliper. Individual egg shell was air-dried in egg crates for a week and the relative shell weight was determined by relating the shell weight to the weight of the egg. Shell thickness was measured to the nearest 0.01mm using a micrometer screw gauge. The egg length was measured as the distance between the broad and narrow ends of the egg while the width was measured as the distance between two ends of the egg at the widest cross sectional region using vernier calipers.
Internal quality of eggs Eggs were broken onto a glass-topped table; the albumen height (AH) was measured with a tripod micrometer at its widest part at a position halfway between the yolk and the outer margin. Albumen weight was taken as the difference between the egg weight and the sum of the weight of the yolk and dry egg shell while the percentage albumen weight was calculated as the percentage of the albumen weight to the egg weight. Yolk weight was measured using Mettler All data collected were subjected to One-way Analysis of Variance in a 2 x 3 factorial arrangement using SAS software (SAS, 2000) while signi cant (p<0.05) means among variables were separated using Tukey's HSD.

Main effects of phytobiotic type and inclusion levels on laying performance indices
The main effects of phytobiotic type and inclusion levels on laying performance indices are presented in Table 2. Henday egg production, hen-housed egg production, egg mass and feed conversion ratio were signi cantly (p<0.05) in uenced by phytobiotics type. Hen-day production and hen-housed production of laying hens fed dietary ginger were signi cantly (p<0.05) higher than those fed turmeric powder rations. Birds placed on ginger powder ration had a higher (p<0.05) egg mass of than those of turmeric powder.  Inclusion levels of phytobiotics did not signi cantly (p>0.05) impact the hen-day production but hen-housed egg production was positively in uenced (p<0.05) with birds on 1.5% and 3% inclusion levels higher than those on 0% inclusion levels. Percentage mortality of laying birds fed 1.5% inclusion levels were signi cantly lower compared to those on 0% inclusion level. The earliest age (p>0.05) at rst lay (179.13days) and the highest weight (p<0.05) at rst lay (1745.00g) were observed among birds fed 3% inclusion level of phytobiotics. Weight at rst lay in pullets fed ginger diets was numerically higher than those offered turmeric powder.

Interactive effects of phytobiotic type and inclusion levels on laying performance indices
The interactive effects of phytobiotic type and inclusion levels on laying performance indices are presented in Table 3.
The interaction between phytobiotics and inclusion levels in uenced (p<0.05) hen-day egg production, hen-housed egg production, egg mass and FCR. Hen-day egg production of hens fed dietary inclusion levels of ginger powder at 0% (60.73%) and 1.5% (62.26%) were higher (p<0.05) compared to values obtained for hens fed diets containing 0% (52.97%) and 1.5% (53.62%) turmeric powder. Hen-housed was highest among birds placed on ginger powder diets at 1.5% inclusion levels which recorded 62.26%, although not statistically different from those fed ginger and turmeric Loading [MathJax]/jax/output/CommonHTML/jax.js powder at 3%, while the lowest value of 49.53% was noted for those fed 0% turmeric powder diets. The highest value obtained for egg mass among birds fed 1.5% ginger powder diets was 46.14g/hen/day while those on dietary inclusion of turmeric powder diets recorded the least value of 36.11, 35.34 and 36.55g/hen/day. The least FCR value of 2.64 was obtained in hens fed gingerised diets at 5% dietary inclusion while hens maintained on 1.5% dietary turmeric recorded the highest values of 4.00. Egg weight and feed intake were not in uenced (p>0.05) by the interaction between phytobiotic type and inclusion levels. Mortality was the highest (p>0.05) among birds supplied 0% ginger powder ration (14.63%), followed by birds on 0% turmeric powder ration while the least value of 1.92% was obtained each from birds fed 1.5% ginger and 1.5% turmeric powder diets. Birds offered 1.5% turmeric powder attained lay much later than the rest of the treatment groups (192.75days). Age at rst lay was attained earliest (p<0.05) by pullet birds placed on 1.5% ginger powder diets recording 166.25 days while birds fed turmeric powder diets attained lay at later age of 192.75 days. The highest weight (p<0.05) at rst lay was observed among those offered 3% ginger powder diets (1805.00g) while pullet birds which consumed the control diets (1595.00g; 1510.00g), those on 1.5% ginger (1507.50g) and 1.5% turmeric (1537.50g) powder rations recorded the lowest weight at rst lay.  Haugh unit (91.54) noted for turmeric powder group. However, birds fed diets containing ginger powder had thicker shell (p<0.05) of 0.46mm in while a lower value of 0.44mm was observed in hens placed on turmeric powder diets. Interactive effects of phytobiotic type and inclusion levels on egg quality traits of pullets The interactive effects of phytobiotic type and inclusion levels on egg quality traits of pullets are presented in Table 5. All parameters measured except egg length, egg width, ESI and % albumen weight were signi cantly (p<0.05) impacted by the interaction between the treatment factors. Birds fed 1.5% (6.95g) and 3% (7.00g) dietary ginger powder produced eggs with the highest shell weight while those on 0% ginger powder rations recorded the lowest value of (6.17g). % shell weight (11.06%) was the highest among birds on 3% ginger powder diet while the least was obtained from those fed 0% ginger powder ration. Laying birds on the control groups (0.43 and 0.44mm) and 1.5% dietary turmeric (0.43mm) laid eggs with lower shell thickness while those placed on 1.5 (0.47mm) and 3% (0.48mm) ginger powder diets 0.47 and 0.48mm).

Interactive effects of phytobiotic type and inclusion levels on haematological indices of laying pullets
The interactive effects of phytobiotic type and inclusion levels on haematological indices of laying pullets are presented in Table 7. All the parameters measured were not signi cantly (p>0.05) in uenced by the interaction between phytobiotic type and levels of inclusion. PCV haemoglobin, WBC heterophils, MCV, MCH, and MCHC values increased as ginger powder inclusion level increased while the trend differed among birds fed turmeric powder diets. Birds on 3% turmeric powder diets recorded the highest values of PCV (36.26%), haemoglobin (11.85g/dl), RBC   Lower mortality observed among the phytobiotic groups compared to the control groups (11.71 and 9.82%) was probably due to greater capacity of the supplied ginger and turmeric powder rations to reduce morbidity among the birds as a result of their medicinal functions (Chattopadhyay et al., 2004), culminating in improved livability. Previous studies conducted using phytobiotics as feed additives have shown encouraging results with respect to lowered mortality and increased livability in poultry birds (Issa, 2012 and Oleforuh-Okoleh et al., 2014). Hertog et al. (1993) and Knekt et al. 1996) reported that the phenolic avonoids constituents of phytobiotics have been shown to have an inverse association with morbidity and mortality.
Besides this, turmeric has been noted to ameliorate the effects of a atoxin, common in maize-based feed, causing hepatotoxic and hepatocarcinogenic effects, which poses serious challenge to poultry production (da Rocha et al. Conversely, high mortality rate experienced among the control groups (11.71 and 9.82%) in the present study could partly be due to the hot environmental conditions at some period (between January and March, 2018) of the experiment leading to heat stress in the laying birds. High ambient temperature or heat stress has been a major environmental stressor and thus a concern for the poultry industry, mostly in the hot regions of the world. Growth, feed intake and egg production are usually negatively in uenced (Haruhiko et al., 2015) during heat stress. Good management which includes good control of feeding timetable, lighting programme (ISA, 2009) as well as nutritional strategies aimed at eradicating the negative in uence of heat stress through the use of medicinal herbs (such as ginger and turmeric), and micronutrients like vitamins and minerals to meet the requirements of birds during heat stress have proven to be of immense advantage (Lin et al., 2006). According to Devegowda (1996), curcumin present in turmeric are included in feed for the enhancement of animal performance by improving livability and lowering mortality in poultry birds.
The attainment of lay at an earlier age among birds fed ginger powder rations (as opposed to a later age obtained for the turmeric groups) could possibly be connected to higher body weight at rst lay among these groups. The earliest age at rst lay observed among the birds placed on 1.5% ginger powder group is higher than those obtained by Islam et al. (2015) and Kabir and Haque (2010) who both recorded 141days in Isa Brown pullets. Age at sexual maturity is a very vital trait from economic standpoint. The age at rst oviposition is crucial, not only because it determines its rst year production but also because the earlier a pullet comes to lay, the sooner revenue is generated (Jull, 1970). However, the least age at rst lay obtained in this research was older than the recommended (126days) for ISA Brown by Hendrix Genetic Company Limited (www.HendrixGenetics.com), and this variation may be due to environmental factors. In most domestic animals, reproductive functions are known to be considerably affected by nutrition (Armstrong and Benoit, 1996;Williams, 1998). Feed consumption, lighting schedule, length of daylight and environmental factors are major factors that determine age and weight at sexual maturity (Morris and Fox, 1960). The commercial layer usually comes to lay between 18 and 19 weeks (126-133 days) of age, egg production then rises sharply to a peak of 94-96% (www.HendrixGenetics.com) at 26-27 weeks of age and nally declines gradually (Rahman, 2003). Age at sexual maturity is also in uenced by pronounced sexual effect (Eaton, 1961) which is characterized by egg lay. Therefore, the probable explanation for this delay may be due to the fact that a threshold of ovary and oviduct weights must be achieved before sexual maturity is attained; since these organs are responsive to feed allocation and in uenced by body weight pro le (Robbinson et al., 2007). The impact of the bioactive compounds of turmeric powder on the reduction of adipocyte number which resulted in decreased fat accretion in uencing body weight and in turn the age at rst lay (Attia, 2018) may as well be responsible for this delayed sexual maturity.
Egg production is the most important index of performance of commercial layer and it accounts for 90% of the income from the enterprise (Oluyemi and Robert, 1979). The results obtained in this study depicted that hens maintained on 1.5% ginger powder diets had superior laying performance in terms of hen-day egg production, henhoused egg production, egg mass and feed conversion ratio. Gingerol compounds contained in ginger powder may be responsible for the improvement in the digestive tract of laying hens which in turn boost egg production. This observation is comparable to those reported by Moeini et al. (2011) who discovered that the incorporation of 1% ginger rhizome powder increased the egg production and egg mass in white leghorn laying birds. Similar values obtained for hen-day and hen-housed production birds on 1.5% ginger powder diets indicated low mortality rate among these birds during the laying period. Abdollah et al. (2011) documented an increase in egg production with 0.5% dietary inclusion of ginger root with no negative effect on egg weight and feed conversion ratio of laying birds. The authors also found that egg production and egg mass in the groups fed diets containing turmeric powder were higher than those of control groups. Samarasinghe et al. (2003) reported that 0.1, 0.2 and 0.3% turmeric treatments did not affect feed intake, egg production, egg weight and egg mass.
The incorporation of phytobiotics at varied inclusion levels did not impact egg weight among the dietary treatment groups. This could be due to the low levels or concentrations of the primary (protein content and fats) compounds and the volatility of the secondary compounds (volatile oils) in ginger and turmeric powder which may be required to enhance egg weight. The nding is similar to Samarasinghe et al. (2003) and Emadi and Kermanshahi (2007) but contrary to Pandian et al. (2013) and Kanagaraju et al. (2016). Malekizadeh et al. (2012) reported that turmeric powder at 1 or 3% inclusion level had no bene cial effect on egg weight and reduced egg production compared to the control groups. The ability to make signi cant changes to egg weight enables the egg producer to adapt to market demands (ISA, 2009), since most consumers tend to appreciate larger egg size. According to ISA (2009), it is possible to change egg size by 1 to 3g by changing the age at sexual maturity, or the body weight at start of lay.
The mean values obtained for egg width and egg shape index (ESI) in the current study differed from the ndings of Attia (2018) who reported that the incorporation of 1.5 and 3% of turmeric powder in the layer diet did not show improvement on ESI among the groups. Nasiroleslami and Torki (2010) reported that addition of essential oil of ginger had no signi cant impact on ESI. The signi cantly higher values of albumen height, albumen weight, % albumen weight noted in hens maintained on 3% turmeric powder suggests that eggs produced by this group are of superior quality compared to the other treatment groups. Increase in albumen showed that the bioactive compounds in turmeric powder stimulated the growth of the epithelial cells and tubular gland cells in the magnum of the reproductive tract to synthesize and secrete substantial amount of albumen compared to the rest of the groups (Saraswati et al., 2013).
Regarding yolk parameters, birds on 3% turmeric powder produced eggs with the best values of yolk weight and yolk colour which implies that turmeric had a major role to play in improving the yolk quality of eggs. Park et al. (2012) observed that the value of yolk colour in Lohmann Brown laying hens fed 0.50% turmeric powder was higher than those in control. Yolk colour is very vital feature which determines the acceptability of the egg and depends on the presence and pro le of carotenoids in feed (Puvača et al., 2018). Jacqueline et al. (1998) reported that the body system of laying hens cannot synthesize egg yolk pigments (Karaskova et al., 2015) by their own biochemical processes. This suggests that the yellow orange pigment present in turmeric was deposited in the yolk. This may also have contributed to the higher yolk weight recorded for this group of birds. Thus, the natural yellow-orange substances in turmeric could be used to improve yolk colour in light-coloured feeds (Park et al., 2012). According to Riasi et al. (2012), enhancement of yolk color could be as a result of curcumin, curcuminoids and its related compounds which are the yellowish pigment of turmeric.
Consumers give preference to deep yolk colour (Englmaierova et al., 2014) as they are associated to healthier and more natural eggs. Therefore, some phytobiotic additives are commonly added to laying hen diets to enrich the yolk. Xanthophyll is attached to fat-soluble pigments in the feed (Yıldırım et al., 2013). Carotenoids modify egg yolk colour and are a source of red and yellow (xanthophylls) pigments (Englmaierova et al., 2014).
The higher Haugh unit of birds placed on 1.5% turmeric powder in the present study could mean that turmeric enhanced the internal quality of the eggs produced by this treatment group and thus may prolong the keeping quality during storage. Our result was higher than those obtained by Nasiroleslami and Torki (2010) who reported a value of 70.64. The Haugh unit is the most widely accepted measure of internal egg quality and tends to decrease with the time of storage (Williams, 1992). The quality of eggs and their stability during storage are largely determined by their physical structure and chemical composition (Seidler, 2003).
The results of shell weight (7.00g), % shell weight (11.06%) and shell thickness (0.48mm) obtained in 3% dietary ginger groups revealed that eggs produced were of superior external quality. These values are in agreement with those of Zomrawi et al. (2014) who observed higher values of shell weight and thickness in laying hens fed varying levels of ginger root powder. The use of ginger in layer diets might have caused an improved environment in the shell gland (uterus), which is a calcium deposition site, and thus enhanced shell weight and thickness (Radwan et al., 2008). The observation in the present study is similar to the ndings of Nasiroleslami and Torki (2010) who concluded that addition of the essential oil of ginger increased egg shell weight and thickness in laying hens. In contrast, Incharoen and Yamauchi (2009) reported there was no difference in shell thickness and shell ratio with 1 and 5% dietary dried fermented ginger. The relationship observed between egg shell thickness and ESI in the current study is in conformity with the ndings of Altuntaş and Şekeroğlu (2008), who reported that the mean shell thickness increased as ESI increased.
Although, all haematological parameters measured in this current study were not signi cantly in uenced by dietary ginger or turmeric, yet, the slight increase in PCV and Hb levels in birds placed on turmeric diets may indicate an enhanced oxygen carrying capacity of the blood (Larsson et al. 1985), and could be linked to the elevation observed in the red blood cells. Increased RBC depicts a better transportation of red blood components in response to erythropoietic system stimulation which may be associated with the effects of bioactive compounds in turmeric to improve the antioxidant status of the birds (Rababah et al., 2004). Afolabi (2010) posited that haematological parameters of farm animals are affected by a number of factors: breed, climate, geographical location, season, day length, time of day, nutritional status, life habit of species, present status of individual among other factors. MCV, MCH and MCHC levels obtained in this study were in line with the normal range (90-140fL, 33-47pg and 26-35g/dl respectively) as reported by Patra et al. (2010). A low level of MCH and MCHC is an indication of anaemia (Aster, 2004) in farm animals and humans. It is noteworthy that the normal levels of these parameters among birds placed on turmeric powder diets could mean that turmeric inclusion in the diet did not negatively impair the transport of oxygen in the body tissues via the blood throughout their laying period. According to Oyawoye and Ogunkunle (2004), haematological components such as MCV, MCH and MCHC are valuable in monitoring feed toxicity especially with feed constituents that affect the blood as well as the health status of farm animals.

Conclusion
Based on the results of this study, it can be concluded that the effect of ginger and turmeric powder on the performance of pullets depends on their inclusion levels. Moreover, ginger powder enhanced sexual maturity and onset of egg lay in egg-type chickens. Egg-type chickens can be fed ginger and turmeric powder diets up to 3% inclusion levels over their commercial production lifespan in order to enhance their reproductive performance without impairing their overall growth. Laying birds which consumed diets with 1.5% of ginger powder had the best performance in terms of hen-day, hen-housed production, egg mass and FCR.
The external egg qualities (shell weight, % shell weight and egg shell thickness) were more enhanced by ginger powder inclusion while superior internal qualities (albumen height, albumen weight, % albumen weight, yolk colour, and Haugh unit) were obtained from eggs produced by birds raised on turmeric powder diets. The inclusion of 1.5% turmeric powder in layer feed may enhance egg quality due to higher Haugh unit values.
Both ginger and turmeric powder did not impair the health status of the laying birds by stimulating their immune response through increased lymphocytes count.

Con ict of Interest
Authors declare they have no con ict of interest.

Ethics Approval
The procedure used in this study was approved by the Federal University of Agriculture, Abeokuta, using the guideline of the Nigerian Institute of Animal Science (NIAS).

Consent to Participate
Not applicable

Consent for Publication
All authors have given their consent to publish this study.

Availability of Data and Materials
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.