The impact of in ovo feeding with different L- arginine to L- lysine ratios on small intestine histological characteristics and immune system organs in day-old chicks

The avian embryo develops in a carbohydrate free environment and restricted resources of energy and nutrients for supporting its growth (Foye et al. 2006a). In ovo injection is a method for supplying nutrients into amniotic fluid, which further can be ingested orally and improves intestinal development and function (Uni et al. 2005; Foye et al. 2007). Previous studies showed positive effects of arginine and lysine on intestinal growth and morphology (Foye et al. 2006 b; Nayak et al. 2016; Ebrahimi et al. 2017). Al-Murrani (1982) reported that 85.7% L- arginine to L- lysine ratio is adequate for growth of 7 d-old chick embryo. Though there are few reports regarding the effect of different L- arginine to L- lysine ratios on chick embryo intestine growth. Therefore, the aim of this study was to evaluate the effect of in ovo injection with different L- arginine to L- lysine ratios on small intestine growth and its morphology, and also the immune system organs’ weight of a day-old Ross 308 broiler chicks. Material and A total of 210 fertile Ross × Ross 308 broiler eggs were purchased from a breeder flock with 24 weeks of age. Eggs were weighed and then assigned into 7 experimental groups with 30 individual eggs per each group. Treatment groups were included: non-injected control, shamcontrol (distilled water), and 75.7, 80.7, 85.7, 90.7, and 95.7 % L- arginine to L- lysine ratios, which was injected into the amniotic fluid on 14 d of incubation from the broad end of eggs. Lysine was considered 2% based on a previous experiment (Ebrahimi et al. 2017) and then, L- arginine to L- lysine ratios were considered based on what Al-Murrani (1982) reported for 7 d old chick embryo with 5% higher and lower intervals (85.7%). On d 22, chicks were weighed and slaughtered. Afterward, length and weight of duodenum, jejunum, and ileum were recorded. Also, thymus (right lobes), bursa of Fabricius, and spleen were weighed. Moreover, relative weight of these organs to hatchling weight was calculated. For intestine morphology evaluation, segments of duodenum, jejunum, and ileum drowned in 10% neutral buffered formalin around one week for fixation. After fixation, tissue samples were processed using dehydration protocol (series of graded alcohols), clearance by xylene, and embedment in paraffin. Then, these samples were cut into 5 µm thick cross sections using microtome instrument and were mounted on slides. Then, staining process was performed using Hematoxylin and Eosin (Poosti and Adibmoradi 2008). Finally, villus height, crypt depth, villus width and crypt diameter were measured by a Zeiss light microscope. Data were then analyzed based on a completely randomized design by the GLM procedure of SAS 9.2 software. Based on the results, in ovo feeding of different L- arginine to L- lysine ratios up to 85.7% had an increasing effect on chick weight (P<0.01; Table 1). Similar to the present results, Edwards et al. (2012) and Nayak et al. (2016) reported that in ovo injection of arginine had an increasing effect on chick weight. Also, it was indicated that in ovo injection of lysine improved hatchling weight (Asmawat et al. 2015; Ebrahimi et al. 2017). The improving effect of different L- arginine to L- lysine ratios partly may be mediated by growth stimulatory effects of arginine (stimulating secretion of insulin and growth hormone along with increasing synthesis of nitric oxide and polyamines), (Floyd et al. 1966; Davis et al. 1972; Jobgen et al. 2006; Khajali and Widerman 2010) and partly by growth stimulatory effects of lysine (stimulating carnitine synthesis and as a result, increasing IGF-I secretion), (Arslan et al. 2004; Kita et al. 2002). Treatments also increased bursa of Fabricius weight (P<0.05) and the highest amount was observed by 85.7% L- arginine to L- lysine ratio (Table 1). In accordance with the present study, in ovo injection of lysine up to 20 mg improved bursa of Fabricius weight (Ebrahimi et al. 2017). In ovo injection of different L- arginine to L- lysine ratios had an increasing effect on jejunum (P<0.01), ileum (P<0.05), and small intestine length (P<0.01), relative weight of small intestine to chick live body weight (P<0.01), and duodenum, jejunum, and ileum weights (P<0.05; Table 2). Also morphological results indicated higher villus height and villus height/crypt depth ratio, while lower crypt depth in jejunum and ileum by almost all treatments in comparison with control groups (Table 2). Also, 90.7% L- arginine to L- lysine ratio had the best improving effect on the most of intestinal parameters. Similar to the present results, Edwards et al. (2016) showed that in ovo injection of arginine improved weight, length and villus height of intestine in chicks. Foye et al. (2007) indicated improving effect of in ovo injection with arginine on digestion and absorption capacity of jejunum in turkey. Ebrahimi et al. (2017) reported that in ovo injection of 20 mg L- lysine increased (P<0.05) jejunum weight and also villus height, villus height/crypt depth ratio, while reduced crypt depth of duodenum, jejunum, and ileum. As increasing in intestine length, villus height, and villus height/crypt depth ratio are indicators for high nutrient digestion and absorption, it may be concluded that in ovo injection of 90.7% L- arginine to L- lysine ratio will be able to improve growth of chicks during the rearing period (Pluske et al. 1997; Caspary 1992; Ebrahimi et al. 2016, 2017). Improving effect of arginine may be mediated by activating mTOR signaling pathway which resulted in higher protein synthesis, while lower protein degradation (Tan et al. 2010). Lysine may improve intestinal growth parameters by biosynthesis of L-carnitine and then stimulating IGF-I secretion which can result in improvement of intestine growth (Arslan 2006; Shafey et al. 2010). In the present study, though 85.7% L- arginine to L- lysine ratio had the best effect on hatchling and bursa of Fabricius weight, in ovo injection of 90.7% L- arginine to L- lysine ratio had the best improving effect on weight, length, and morphological parameters of broiler chicks, then is a suggestible level for in ovo injection.