Glycosaminoglycans and vitamin C in ovo feeding affects bone characteristics of chicks

Different levels of glycosaminoglycans (chondroitin sulfate and glucosamine sulfate) and vitamin C were injected in ovo to determine whether additive use influences the incubation parameters and bone characteristics of posthatch chicks. Hatchability was not affected by addition of glycosaminoglycans and vitamin C in ovo. However, chicks that received 4 μg additive showed 2.86% reduction in total mortality when compared with chicks from non-injected eggs. Moreover, tibia area and femur bone mineral density increased in chicks from eggs injected with 2.16 and 6.00 μg of additive, respectively. In ovo feeding with glycosaminoglycans and vitamin C can benefit bone development in embryos and reduce total mortality during the incubation period.


Introduction
Upon reaching rapid body growth, efficient feed utilization, and maximum breast meat deposition, broilers begin to show disorders in their bone structure, with increased occurrence of angular and torsional deformities, tibial dyschondroplasia, rickets, osteochondrosis, and femoral necrosis (Almeida Paz et al., 2010).Bone deformities and fractures are severe health problems in modern, rapidly growing broilers, because genetic breeding programs aimed at superior performance have not included improvements in bone development (Almeida Paz et al., 2010).
In ovo feeding can be regarded as supplemental nutrition for the embryo and yolk sac (Santos et al., 2010), which may benefit embryo development and quality of posthatch chicks (Uni and Ferket, 2004).
Polysulfated glycosaminoglycans (GAG) such as glucosamine and chondroitin have anti-inflammatory action and are pharmacologically classified as slow-acting symptomatic drugs that reduce the symptoms of bone disorders such as osteoarthritis in humans and some animal species (Brandt et al., 2002).Additionally, they stimulate the synthesis of proteoglycans and collagen, increasing the proliferation of chondrocytes and bone matrix biosynthesis (Altman et al., 1989;Clark, 1991).
Vitamin C, or ascorbic acid, participates in several biological processes.It helps in collagen synthesis and maintenance, is essential for the formation and maintenance of connective tissue, bone, and cartilage, and stimulates the immune system, increasing disease resistance (Berzina et al., 2013;Sgavioli et al., 2013;Chand et al., 2014).
Considering the existing research results about the benefits of vitamin C supplementation in broilers and chondroitin and glucosamine sulfates in other animal species, this study was conducted to evaluate the use of GAG and vitamin C in ovo feeding on bone system of developing posthatch chicks.

Material and Methods
This study was approved by the local Committee of Ethics in Animal Use (case no.011424/13) in Jaboticabal, SP, Brazil (21º14'05" South latitude and 48º17'09" West longitude, and average altitude of 615.01 m).
Eggs were distributed into five treatments, in a completely randomized experimental design, as follows: eggs non-injected (unperforated) and eggs injected with 0, 2, 4, or 6 μg additive/100 μL water on the fourth day of incubation.
According to Shim and Pesti (2011), in broiler embryos, the bone system starts to develop on the fourth day of incubation; for this reason, this date was chosen.Each 100 g of additive contained 30 g chondroitin sulfate, 30 g glucosamine, 5 g vitamin C (Synth, 99% purity, Diadema, Brazil), and 100 g excipient q.s.The chondroitin sulfate [(C 14 H 21 NO 14 S)n, Infiniti Nutraceuticals, Inc.] had a purity of 91.35%, and the potassium glucosamine sulfate [(C 6 H 14 NO 5 ) 2 SO 4 × 2KCl, Zheijiang Freemen Inc.] had a sulfate content of 15.7%.
The formulation of the additive injected in ovo was based on the veterinary medicine Condrodoton 500 μg (Vetnil ® , Louveira, São Paulo, Brazil), recommended for dogs.The supplied quantity of chondroitin sulfate, glucosamine sulfate, and vitamin C in micrograms were thus converted according to the dog weight in pounds to the quantity in micrograms of those substances for broiler weight in pounds.Results of studies on broiler nutrition (Ferreira et al., 2015;Sgavioli et al., 2017) were also used to adjust the glucosamine and chondroitin doses in the additive injected.
For the additive injection, the eggs were held horizontally and, after cleaning with 70% ethanol, the shell was perforated near the thin end (the end opposite to the air cell) with a sterile needle [Injex,13 × 0.38 (27.5 G1/2")] through which 100 μL aqueous additive solution were injected into the albumen, approximately 6 mm below the membrane.The solution was prepared with ultra-pure water autoclaved in the dark because of its photosensitivity (Sgavioli et al., 2013).After injection, the hole was sealed with a label identifying the treatment and replicate.
The relative weight (%) of chicks post-hatch was obtained as the absolute weight of the chick (g) relative to the egg weight (g) (at day 0 of incubation).
Egg mass loss was calculated as the difference in egg weight before incubation and weight at the 18th day of incubation, expressed as a percentage of initial egg weight.Eggshell conductance was calculated as egg mass loss (g) divided by steam saturation pressure (23.86 mm/Hg at 25 ºC).
Eight birds per treatment whose average body weight was close to the average weight of the experimental unit were sacrificed at one day of age, in a total of 40 animals.Birds were stunned by individual exposure to CO 2 gas for 2 min in a flow-through system and then slaughtered by cervical dislocation.
Bone mineral density (BMD) (g/cm 2 ), area (cm 2 ), and length were determined in the left femur and tibia using dual-energy X-ray absorptiometry (Horizon Discovery Dxa Hologic ® , Massachusetts, USA).Prior to sample scanning, the device was calibrated with a phantom, supplied by the manufacturer (Hologic ® , Massachusetts, USA), as bones.For all analyses, a small-animal software (Hologic ® , Massachusetts, USA) was used.Clean bones were placed with the cranial face in contact with an acrylic container with deionized water and scanned using a densitometer.The small-animal software was used to select the region for subsequent densitometric analysis.After the densitometric analysis, the bone length was assessed using a digital micrometer (MDC-Lite, 0.001 mm resolution, Mitutoyo, Suzano, SP, Brazil).
Left femur and tibia were used for the mechanical bone-strength tests (three-point bending).The tests were conducted using an EMIC ® (DL 3000) universal testing machine.The load was applied at a rate of 5 mm/min with a force of 2000 N to determine the maximum permissible force (Fmax) of the bone and the deformation (bend) caused by Fmax.Bones were fixed on two supports (two points), with span adjusted according to the size of the smallest bone.The force was then applied at the geometric mean point of the bones between the two supports (the middle third of the bone), and the equipment recorded the results.These variables express bone strength at the ends and in the middle third of the bone.
Right femur and tibia were used to determine bone calcium, phosphorus, and ash contents.The soft tissue was removed, and bones were boiled in deionized water for 5 min.After drying at room temperature, samples were immersed in petroleum ether for 48 h, dried in a forcedventilation oven at 60 ºC for 48 h, and then ground in a ball mill.Bone mineral content was determined using wet analyses.Ash content was determined by burning the samples at 600 ºC.The methods were applied according to Silva and Queiroz (2002) and expressed as a percentage of defatted dry matter or mineral matter.
The effects of incubation treatments (eggs non-injected and injected with 0, 2, 4, or 6 μg additive) on all studied parameters were analyzed statistically using the model described below: in which Y = studied parameter; µ = mean value of the parameter; Ti = treatment (0, 2, 4, or 6 μg additive); and eij = residual error.
Data were subjected to analysis of variance by the General Linear Model procedure (GLM) of SAS ® (Statistical Analysis System, version 9.2) and analyzed for the presence of outliers (Box-and-Whisker plot), normal distribution of studentized errors (Cramer-Von-Mises test), and homogeneity of variances (Brown-Forsythe) (Littell et al., 2006).
Means were compared by 5% probability polynomial orthogonal contrasts, as follows: contrast 1 -comparison between the non-injected egg treatment versus the average of treatments with 0, 2, 4, and 6 µg of additives injected; contrasts 2 and 3 -comparisons using linear and quadratic regression models (0, 2, 4, and 6 µg of additive) (Robbins et al., 1979) to check the effects of additive application.
For embryonic mortality, data were analyzed for frequency by Fisher's exact test at the 5% probability level.

Results
Hatchability, egg mass loss, conductance, chick relative weight (Table 1), and embryonic mortality (Table 2) did not differ based on the analyzed contrasts (P>0.05).
In the embryo diagnosis, there was an effect for intermediate (P = 0.0083), late (P = 0.0083), or total (P = 0.0167) mortality, with the lowest mortality found in the treatments injected with 0, 2, and 4 µg additive, respectively (Table 2).
There was a contrast-1 effect (non-injected eggs vs. injected eggs) for the femur area (P = 0.0175).Smaller femur areas were observed in injected eggs (0.37 cm 2 ) when compared with non-injected eggs (0.40 cm 2 ) (Table 3).  1 Additive composition: each 100 mg of additive contained 30 mg of chondroitin sulfate, 30 mg of glucosamine, and 5 mg of vitamin C (excipient q.s.-100 g).Probability: P>0.05. 2 Egg mass loss: difference in egg weight before incubation and at the 18th day of incubation, expressed as a percentage of initial egg weight. 3Relative weight: absolute weight (g) of the chicks relative to egg weight (g) (at day 0 of incubation). 4Treatments used for the regression calculation: 0, 2, 4, and 6 µg of additive.
The maximum strength and deformation of the femur did not differ based on the analyzed contrasts (P>0.05)(Table 4).
Femur and tibia concentration of phosphorus (P) responded linearly (P = 0.0343 and P = 0.0353, respectively) for all injection levels (Table 5).According to the equations P tibia = −0.112additive + 18.271 (R 2 = 0.78) and P femur = −0.106additive + 19.358 (R 2 = 0.95), when the additive injection concentration increases, there is a reduction in the phosphorus concentration of the femur and tibia of chicks.

Discussion
In ovo feeding with chondroitin sulfate, glucosamine sulfate, and vitamin C was evaluated in terms of ability to promote changes in bone and cartilage development posthatch.Despite the increase in femur and tibia area and femur BMD observed with in ovo feeding (GAG + vitamin C), the phosphorus concentration in the femur and tibia declined as the injected concentrations of in ovo feeding were elevated.
The maximum derivative of equations for tibia area and femur BMD revealed that in ovo feeding with 2.16 and 6 µg of additive maximizes the response to the cited characteristics, respectively.
Glycosaminoglycans and vitamin C can influence the formation of long bones, as these substances stimulate the synthesis of the proteoglycans and collagen and are capable of increasing the proliferation of the chondrocytes and biosynthesis of the matrix (Clark, 1991;Sgavioli et al., 2017).They are essential in the endochondral ossification process, responsible for the longitudinal growth of long bones, due to epiphyseal disk calcification (Anderson et al., 2005).0.5069 0.5261 0.0333 3 0.7487 0.0315 3 0.5339 SEM -standard error of the mean; CV -coefficient of variation.
1 Additive composition: each 100 mg of additive contained 30 mg of chondroitin sulfate, 30 mg of glucosamine, and 5 mg of vitamin C (excipient q.s.-100 g). 2 Treatments used for the regression calculation: 0, 2, 4, and 6 µg of additive. 3Significant at P<0.05. 1 Additive composition: each 100 mg of additive contained 30 mg of chondroitin sulfate, 30 mg of glucosamine, and 5 mg of vitamin C (excipient q.s.-100 g). 2 Treatments used for the regression calculation: 0, 2, 4, and 6 µg of additive.There are several studies on the effects of in ovo feeding with injection of vitamin C (Ghonim et al., 2009;Mohammed et al., 2011;Nowaczewski et al., 2012) in broilers.However, scientific papers that correlate vitamin C in ovo feeding and bone development in broilers are scarce.Sgavioli et al. (2015) injected 6 μg ascorbic acid in ovo before incubation and did not observe beneficial effects on bone development (femur and tibia densitometry, mineral density, and bone strength) of birds at 42 days of age.
Therefore, our hypothesis is that in ovo feeding with GAG may be more effective in promoting bone development of posthatch chicks than vitamin C.However, to confirm this hypothesis, further research should be conducted to evaluate the isolated effects of each additive on the bone development of birds.
Higher phosphorus concentration in the femur and tibia were observed when the eggs were injected with water only; therefore, inclusion of GAG and vitamin C linearly reduces the concentration of phosphorus in the analyzed bones.A lower percentage of bone phosphorus can influence bone mineralization and affect bone mechanical quality, in broilers.According to Vargas Junior et al. ( 2004), calcium and phosphorus are the most essential minerals for bone formation, since 98% of the calcium and 80% of the phosphorus in the body are found in bones.However, the lower tibia and femur phosphorus mineral composition did not affect the density or bone-strength parameters.
According to Uni and Ferket (2003), high concentrations of solutions can interfere with osmotic equilibrium and affect embryo development.These authors described that the maximum osmolarity of the solution to be injected in ovo is 800 mOsm.The solution applied in ovo in the present study has a lower osmolarity than this limit (86 mOsm).
The lack of treatment effects on initial mortality indicates that in ovo feeding with GAG and vitamin C at the fourth day of incubation did not result in compromised embryo development.Moreover, positive results for total mortality were obtained with in ovo feeding with 4 μg of additive (2.86% mortality reduction when compared with the unperforated treatment).This is unprecedented information, because it shows for the first time in the literature that in ovo feeding with GAG and vitamin C can reduce embryo mortality.
However, Sgavioli et al. (2015) reported a decrease in egg hatchability after injecting increasing levels of vitamin C before incubation.This shows that the effect of in ovo feeding on hatchability varies with the concentration of the solution, stage of embryonic development, and egg region where the solution was injected.Jochemsen and Jeurissen (2002) asserted that the in ovo injection site can be determined according to egg incubation day.According to Ohta et al. (2001), in ovo injection of products must be performed in the extra embryo cavity or yolk sac at the 7th day of incubation to avoid decreased hatchability and to benefit embryo development.

Conclusions
In ovo feeding with glycosaminoglycans and vitamin C can change the bone development of chicks and reduce their mortality.Our research testing in ovo feeding with glycosaminoglycans and vitamin C has established a new science of neonatal nutrition.

Table 1 -
Glycosaminoglycans (chondroitin sulfate + glucosamine sulfate) and vitamin C in ovo feeding on hatchability, egg mass loss, eggshell conductance, and relative chick weight SEM -standard error of the mean; CV -coefficient of variation.

Table 3 -
Glycosaminoglycans (chondroitin sulfate + glucosamine sulfate) and vitamin C in ovo feeding on femur and tibia length, bone area, and bone mineral density (BMD) of posthatch chicks

Table 4 -
Glycosaminoglycans (chondroitin sulfate + glucosamine sulfate) and vitamin C in ovo feeding on femur and tibia maximum force and deformation of posthatch chicks