EFFECT OF DIETARY SUPPLEMENTATION OF THOS (AQUEOUS EXTRACT OF THONNINGIA SAGUINEA) ON CARCASS CHARACTERISTICS AND ON SOME PHYSICOCHEMICAL PARAMETERS IN PECTORAL AND THIGH MUSCLES OF ISA BROWN LAYING HENS

Yao Konan Antoine 1,3 , N’dri Aya Lydie 2 , Konan Kouakou Séverin 4 , Toure Alassane 3 , Coulibaly M’bétiégué 3 , and N’Guessan Jean David 1 . 1. Biochemical Pharmacodynamy Laboratory, Biosciences Department, Félix Houphouët Boigny University of Cocody, PO Box 582, Abidjan 22, Côte d’Ivoire. 2. Laboratory of Genetic and Bioresources Improvement, Research and Training UnityUniversity of Nangui Abrogoua, 02 BP 801 Abidjan 02, Côte d’Ivoire. 3. LANADA/Central Veterinary Laboratory of Bingerville, Po Box: 206, Côte d'Ivoire. 4. Biochemistry department, Peleforo Gon Coulibaly University of Korhogo, BP 1328, Korhogo. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Recently, the poultry industry began to face multi-resistant germs against antibiotics. Therefore, the search of new plant molecules to solve the issue of multi-resistant germs in poultry as an alternative to antibiotics is an imperative. THOS, the aqueous extract of the inflorescence of Thonningia sanguinea revealed efficient antimicrobial, anti-coccidiosis, anti-salmonellosis and antioxidant activity in laying hens. The present study was conducted to assess the effect of THOS on carcass characteristics and on some physicochemical parameters of laying hens pectoral and thigh muscles. For this purpose, 220 Isa Brown one day aged hens were breed in standard condition till 18 weeks old (1113±90g lively mass). Then hens were randomly divided into two (02) groups of 110 hens each. Hens of group II (treated group) received THOS at a rate of 10g/L of water for a week (07 days) while hens of group I (control group) received only water without any additives. At 72 weeks old, six hens were euthanized in each group. Carcass characteristics, post mortem pH, pectoral and thigh meat color, drip loss, cooking loss, moisture, ashes and total mineral rate were evaluated. THOS didn't affect the major carcass characteristics but significantly increased gizzard weight (32±1.14 vs 25±1.6g; p<0.01) and reduced abdominal fat weight (42±14 vs 65±15 g) as compare to control group. pH pm ; drip loss; cooking loss; moisture and ash were not significantly affected by THOS. In the other hand, THOS significantly reduced the redness (a*) of pectoralis major and minor muscles when compared to control group (4.6±0.08 vs 5.2±0.11; p<0.05). The rate of total mineral of thigh muscle was also significantly enhanced by THOS (4.62±0.12 vs 4.16±0.07 g/100g; p<0.05). These results reveal that the use of THOS against multi-resistant germs in poultry could also help enhanced poultry meat quality.
In sub-Saharan Africa, Senegal and Côte d'Ivoire stand out. Indeed, for many years, Ivorian government has set up management structures that have boosted livestock evolution in general and poultry farming in particular. Poultry meat production increased from 9.669 tonnes to 44.451 tonnes from 2001 to 2015, an increase of about 360%. This resulted in a 73% reduction in poultry meat imports from 2.212 tonnes in 2001 to 593 tonnes in 2015 (Anonyme 1, 2016). Nevertheless, the evolution of Ivorian poultry farming, like its sub-region counterparts, is threatened by the appearance of multi-resistant strains due to abusive use of antibiotics used to treat coccidiosis, salmonellosis and avian viruses (Li et al., 2005). Henceforth, research was redirected to plant and spice extracts and essential oils as food additives. These natural substances have proved to be an alternative to solve multi-resistance problem (Christaki et al., 2004).
The aqueous extract of Thonningia sanguinea named as THOS, a parasitic plant, is part of these substances. In the regard of these properties, the use of THOS as poultry food additive, would be an alternative for improving productivity of poultry farms. However, it is important to not lose sight of poultry and consumers safety even though the recent study of Yao et al., (2016) who revealed that THOS is well tolerated by hens' organism. Therefore, the present study was conducted to evaluate the effect of aqueous extract of Thonningia sanguinea(THOS) on carcass characteristics and on some physicochemical parameters in pectoral and thigh meat of Isa Brown laying hens.

Materials:-Vegetal material:-
The plant material is composed of Thonningia sanguinea (balanophoraceae) inflorescences obtained from Yamoussoukro's region (central Côte d'Ivoire). Thonningia sanguinea is a perennial herb plant like. It is also a nonspecific and exclusive parasite of various plants roots. The thickened tubers and rhizomes are commonly sold in local markets for medicinal purposes. Thonningia sanguinea present throughout the humid forest zone of Africa. In Côte d'Ivoire, it is found from the coast to Bamoro forest. This plant is available at vendors of medicinal plants at all times but it is abundant in rainy season.

Animals and experimental procedures:-
This experiment was carried out at the animal laboratory of Veterinary Center of Bingerville. All procedures and animal care were conducted according to the Veterinary Center guidelines and the European Commission recommendations of 18 June 2007 on guidelines for the accommodation and care of animals used for experimental and other scientific purposes.
Two hundred and twenty (220) laying hens were used in this study. 220, one day old (35±3g lively weight) hens were bought from Ivoire Poussins a company of SIPRA group (Ivorian company of Animal Production). These chickens were breed in confinement under standard conditions of breeding in the animal house of Veterinary Center of Bingerville, a part of National Agricultural Development Support Laboratory (LANADA). During breeding period, birds were provided with standard food obtained from Ivograin Bingerville Company (food composition is shown on table 1). At 16 weeks old the chickens (1113 ± 90 g lively weight) were randomly divided into two (02) 370 groups of 110 chickens each. In addition to the standard diet (table 1), hens of group II (treated group) received THOS at a rate of 10g/L of water for a week (07 days) while the hens of group I (control group) received only water without any additives. Chickens were given food and water ad libitum. At 72 weeks old, 5 to 7 hens were euthanized in each group according to each experiment.

Methods:-Preparation of the aqueous extract of Thonningia sanguinea (THOS):-
The inflorescences were rid of any impurities by washing, cut into strips and dried in open air away from sunlight. These inflorescences were then pulverized in a blender (IKAMAG-RCT®, Labortechnick, USA). 100g of powder were dissolved in 1 liter of deionized water and homogenized in a blender. The homogenate obtained was drained on a square of fabric and the filtrate was then filtered three times on absorbent cotton and once on whatman filter paper. This second filtrate thus obtained was put to dry in an incubator (45-50 °C) until complete evaporation of the solvent (Zirihi et al., 2003). The resulting powder what represent the aqueous extract (THOS) was put in an airtight jar and kept away from moisture till use.

Carcass characteristics determination method:-
At 72 weeks old, 6 hens from each group of weight close to the average weight of the group were selected then weighed and euthanized. The chickens were manually plucked with warm water and the carcass weighed before and after evisceration. After this, carcasses were butcher then the pectoralis major and minor muscles, thigh muscles, head, legs, wings, liver, heart, gizzard, intestine and abdominal fat were individually weighed. The yield of each organ in relation to the edible carcass weight (eviscerated carcass) was then determined using the following formula: Pectoral and thigh muscles color determination method:-At 72 weeks old, 6 hens from each group were euthanized. Pectoralis major and minor muscles, thigh muscle and the liver were remove from carcasses. The color of these samples was then recorded on the inner face using a Minolta reflectance colorimeter CR10 (KONICA MINOLTA INC. JAPAN) according to CIE, (1978) color system (L*, a* and b*). Where L* represent luminance or surface lightness; a* redness; and b* yellowness. To accomplish uniformLy the determination of meat color, colorimetry was performed on a fixed portion of breast and thigh meat. Each measure was replicated 3 times on each sample and an average of the 3 measurements was calculated.

Method of post mortem pH (pH pm ):-
Five hens of 72 weeks old were euthanized. Then, the pectoralis major muscles as well as the thigh muscles of each bird were dissected and conserved at 4°C. At different post-mortem times (1h, 24h and 48h), 1g of each sample was collected and grounded with a meat grinder (Moulinex hv2 1300 France). Immediately after grounded, each crushed sample was homogenized in 10 mL of distilled water and the different post mortem pH were determined using a glass electrode pH-meter (Eutech instruments pH 700 Singapour) coupled to a temperature electrode as described by Stewart et al., (1984). Each measure was replicated 3 times on each sample and an average of the 3 measurements was calculated.

Method of cooking loss determination:-
Cooking losses were determined according to the method described by Wood et al. (1981) and revised by Honikel (1998). Five samples of breast and thigh meat were packed in hermetic polystyrene bags. The samples have approximately the same weight and the same geometric shape (e.g. 51.28±3.2g for thighs muscles; 45.32±2.5 for the major muscles and 22.35±1.5g for the minor ones). These samples were then cooked for 15 minutes at 80 °C (temperature corresponding to a core temperature of 75 °C.) in a thermostatically controlled water bath. Then, bags were cooled in water for 45 min and samples were blotted dry with paper towels and reweighed. Cooking losses were the difference of raw weight and cooked weight as shown on the equation below.  ., (1988). Pectoral and thigh muscles were collected from 7 hens euthanized in each group. The sample were immediately weighed and trimmed (for shape and weight uniformity: 100±3g thigh muscle weight; 66±2g pectoral muscle weight). These samples were then packaged in hermetic polystyrene freezer bags containing absorbent paper (paper towels) and stored in tray at 4°C. After 24 hours, the samples were removed from the bags, wiped with absorbent paper and then weighed again. Then the samples were restored again in the previous conditions. This operation was replicated after 48 hours and 72 hours of 4°C storage. Drip losses were expressed as percentage of the initial weight as described in the equation below: Where T is post mortem storage time (24h, 48h or 72h) Moisture determination method:-Pectoral and thigh muscles were ridded of bones and then weighed and dried in a desiccator at 103 °C until a constant mass was obtained. After that, each sample was finely grounded and all the grounded materials obtained were dried again in a desiccator at 103 °C for 35-45 min. After this last drying, the samples were reweighed. The different moisture rates were determined by calculating the variations between the initial and final weight. The results were expressed as a percentage of the raw sample.
Total minerals or ashes determination method:-Total minerals of each sample were determined by calcining dried maters in muffle furnaces at 570 °C for 3 hours in platinum crucibles. It was sometimes necessary to moisten the dried sample once or twice with demineralized water, in order to obtain a whitish ash (Pinta, 1973).
Statistical analyzes:-Data Analyzes were carried out using GraphPad Prism software version 5. The comparison of means two (2) by two (2) was made by Mann-Whitney test. The confidence interval is 95%, the average means were significantly different when P <0.05.

Effect of dietary supplementation of THOS on carcass characteristics:-
The results of THOS dietary supplementation on carcass characteristics are summarized in table2.
The analysis of the results didn't reveal significant differences not only in hens lively weight, but also in slaughtered and in edible carcass weight between both groups of animals. The average weights of pectoralis major and minor muscles, thigh muscles, wings, legs, hearts, liver and intestine were also not significantly different. On the other hand, gizzard average weights were significantly greater in THOS supplemented group than in control group (32±1.4g vs 25±1.6g; p<0.01). The same effect was observed in gizzard yield (2.08% vs 1.64%; p<0.05). Head average weights were also significantly higher in THOS supplemented group than in control group (62±2.3g vs Drip losses (%) = Initial sample weight (g)sample weight at T (g) Sample initial weight (g) x 100 Raw sample weight (g)cooked sample weight (g) = Cooking loss (%) Raw sample weight (g) X 100 372 56±1.2g; p<0.05). In addition, although there were no significant differences, abdominal fat average weight was very low in THOS supplemented group (42±14g) as compared to control group (65 ± 15).
In sum, the incorporation of THOS into the chicken diet did not significantly affect carcass characteristics, but it has significantly increased head and gizzard average weight and yield. This incorporation also resulted in an important reduction of abdominal fat weight even though this reduction was not significant.

Effect of dietary supplementation of THOS on breast and thigh muscles color:-
The results of THOS dietary supplementation on carcass characteristics are summarized in table 3.
The liver color was not significantly different between the two groups of the experiment. But dietary supplementation of THOS significantly reduced the redness of pectoralis major and minor muscles (a* 4.6±0.08 vs 5.2±0.11; p<0.05 and a* 4.4±0.08 vs 4.7±0.05; p<0.05 respectively). Dietary supplementation of THOS also increased the yellowness on thigh muscles on the chickens (9.6±0.11 THOS vs 9.0±0.13 control; p<0.05).

Effect of dietary supplementation of THOS on post mortem pH (pH pm ) evolution:-
The results of THOS dietary supplementation on pH pm evolution are summarized in table 4.
There was no significant difference in the pH pm evolution between the two groups of birds. But comparing pH variation (falling rate), a significant difference was observed in the thigh muscle. In fact, the post mortem pH falling rate was significantly greater in THOS supplemented group than in control group (0. Moreover, regardless of the group and type of muscle considered, there was a significant difference (p <0.01) between the pH pm 1h and 24h and so was the pH pm 1h and 48h. But no significant difference was observed comparing the 24h and 48h pH pm regardless of the group and the muscle type considered.

Effect of dietary supplementation of THOS on drip loss, cooking loss, moisture, dried maters rate (DMR) and total minerals rate (TMR):-
The results of THOS dietary supplementation on drip loss, cooking loss, moisture, dried maters and total minerals summarized in table 5 Regardless the muscle type, no significant difference was observed in drip, cooking loss moisture and dried maters (DMR). But despite, the absence of significant difference, THOS supplemented group thigh muscles lost less water compare to control group thigh muscles.
On the other hand, the total mineral rate (TMR) is significantly higher in the thigh muscle of hens treated with THOS compare to control hens (4.62±0.12 g/100g vs 4.16±0.07 g/100g; p<0.05).

Discussions:-
Laying hen is bred in first intention for its eggs but at the end of laying period, the carcass represents an economic gain for the breeder and an important protein source for consumers. Therefore, the finishing weight is a very important carcass evaluation criterion.
In the present study, statistical analysis revealed no significant difference between the finishing weight of THOS treated and untreated 72 weeks old hens (1938±0.02 g THOS vs 1940±0.03g control). These weights are slightly lower than the average standard weights of the Isa Brown hens at 72 weeks of age which is 1990g.  On the other hand, the lower breast muscles pH u (5.54 ± 0.01 and 5.56 ± 0.01) obtained in the present experiment could be due to cell variability because, according to (Monin, 1988) the pH u differs from one muscle to another and within the same carcass.
The thigh muscle pH pm falling rate was significantly higher in THOS treated group. This difference could be due to ATP hydrolysis acceleration by THOS. According to Bendal, (1973), the falling rate of pH pm is directly proportional to the hydrolysis activity of ATP. Then, any factor modifying ATPase activity results in a similar change in the falling rate of pH pm .
There was no significant difference in cooking loss between the two groups of birds studied, regardless of the type of muscles involved. The losses of 35 g/100g (thigh muscle) and 26±3.5 and 28±0.19 g/100g (pectoral muscles) obtained in our experiment are higher than those reported by De Marchi et al., (2011). This difference could be due to genetic variability because De Marchi et al., (2011) have worked on different strains from those studied in our experiment.
Water losses of the different muscles studied also revealed no significant difference between the two groups of hens. However, water losses are lower in thigh muscle of THOS treated hens than in control hens. This capacity of water retention by the muscles of hens treated with THOS could be due to the presence of polyphenols (N'guessan et al.,  2007), particularly to gallic acid contained in THOS. Indeed, Jung et al. (2010) and Lee et al., (2012) have reported that 1% diet supplementation in gallic acid improves the nutritional quality and water retention capacity of pectoral and thigh muscles of chickens.
Moisture rate obtained in this study (73.45±0.37 to 74.04±0.34 g/100g of muscle) are similar to those reported by Rabot (1998). This author had found moisture rate of 74.7 g/100g for pectoral muscle and 74.2 g/100g for thigh muscle. Moreover, in our experiment, moisture rate of pectoral muscle (73.45 and 73.55 g/100g) doesn't significantly differed from that of thigh muscle (73.62 and 74.04 g/100g). This observation is occurred in the two groups of hens studied. Indeed, in poultry, the moisture rate varied slightly between thigh and the pectoral muscles whatever the breed (BRUNEL et al., 2006).
Unlike the moisture and dried maters (DM) mean rates where no significant difference was observed, thigh muscle total minerals rate (TMR) was significantly higher in THOS treated group. This difference would be due to the reduction in water losses caused by THOS in thigh muscles, which would reduce loss of minerals in the dripping water.

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Regarding meat color, breast muscles redness was significantly lesser in THOS treated hens. All the values of the red index (a * = 4.6 ± 0.08 for the pectoralis major and a * = 4.4 ± 0.08 for the pectoralis minor) obtained in our study are in agreement with that red index value (a * = 4.54 ± 0.14) reported by Chen et al., (2016) in ISA Brownlayers. The low red color observed in breast muscles of THOS treated hens could be due to the difference in fibers types and the amount of pigments such as myoglobin contained in the muscles. Froning, (1995) and Mancini (2009) reported that the red color of meat is related to the amount and the state of oxidation of myoglobin and to the pre-mortem and post mortem conditions. But in our case, the effect of THOS would be a decrease in myoglobin level rather than myoglobin oxidation or degradation because THOS contains many polyphenols. The antioxidant activity of polyphenols improves meat quality, stabilizes the red color of the meat by slowing the oxidation of myoglobin (Krzysztof et al., 2017 and Salami et al., 2017). In addition, the significant pH pm falling rate (0.30) in THOS treated group may also explain the redness loss of breast muscles. In fact, according to Froning, (1995) and Allen et al. (1997), a high rate of pH drop tends to make the meat whiter.

Conclusion:-
The results of the present study show that the use of THOS as Isa Brown laying hens dietary supplementation does not adversely affect the carcass characteristics and the physicochemical parameters of pectoral and thigh muscles. Better, THOS improves gizzard weight and reduces abdominal fat accumulation. This allows hens to value the food consumed. In addition, THOS by its ability to reduce water losses promotes retention of total minerals in chicken muscles. This ability of THOS could improve organoleptic qualities of chicken meat. Finally, THOS reduces pectoral muscles redness. But that isn't a bad outcome when we know that chickens breast muscles are considered as less red meat.
However, other studies should be conducted to elucidate the effect of THOS on pectoral muscles and on organoleptic characteristics of chicken meat in order to better understand the effect of dietary supplementation in THOS on chicken meat quality. Results are expressed as mean ± standard error of the mean (SEM); P = statistical probability NS = no significant difference S* = significant difference (p< 0.05) S** = significant difference (p< 0.01) THOS (10g/L) = aqueous extract of Thonningia sanguinea at the rate of 10 g. l -1 . The slaughter weight is the weight of the bled and plucked subject The edible carcass is the carcass ridded of the viscera, the cloaca; the crop; the proventriculus and the bursa of fabricius The yield of the edible carcass is calculated in relation to the lively weight the yield of the cut of is calculated in relation to the unrefrigerated edible carcass 376 Results are expressed as mean ± standard error of the mean (SEM); P = statistical probability NS = no significant difference S* = significant difference (p< 0.05) THOS (10g/L) = aqueous extract of Thonningia sanguinea at the rate of 10 g. l -1 L* = luminance or lightness of meat a* = red color indicator: (a* < 0 meat seemed more green; a* > 0 meat seemed more red) b* = yellow color indicator: (b* < 0 meat seemed more blue; b* > 0 meat seemed more yellow)  Results are expressed as mean ± standard error of the mean (SEM) Tpm (h) = post mortem time in hour Drip losses; cooking losses; moisture and dried mater are expressed as percentage (g/100g) of raw meat weight TMR = total mineral rate; expressed as percentage (g/100g) of dried maters weight P = statistical probability NS = no significant difference S* = significant difference; p< 0.05 THOS (10g/L) = aqueous extract of Thonningia sanguinea at the rate of 10 g. l -1 DMR= dried maters rate