نشریه پژوهش های علوم دامی
سال بیست و هشتم شماره 2 (تابستان 1397)

Acorn kernel contains a high percentage of starch that can be used as a source of energy in animal’s diet, but it has a high percentage of tannin, which can have deleterious effects on the animals. Tannins are bitter phenolic compounds with a diverse structure that are divided into two groups of condensed and hydrolyzable. Condensed tannins are flavonoid polymers of the proanthocyanidins compounds. Hydrolyzable tannins have a polyhydric alcohol at their core, the hydroxyl groups of which are esterifies with gallic acid. They may have long chains of gallic acid coming from the central glucose core (Reed 1995). Tannins have a high tendency to bind to proteins such as enzymes (Osawa et al. 2000). The harmful consequences of the high percentage of tannin in the diet of ruminants can reduce feed intake, have a negative effect on the activity of rumen bacteria, reduce the efficiency of digestive enzymes in the livestock and also reduce the availability of nutrients, which ultimately reduce growth and production of these animals (Salinger et al. 1996). Also, the high levels of tannin compounds in the poultry diet can reduce feed intake, growth rate, digestion of protein and amino acids, intestinal and pancreatic activity (Armstrong et al. 1974; Brand et al. 1989; Mahmood et al. 1997, 2008). Various chemical and physical methods such as sodium bicarbonate, polyethylene glycol, soaking in water, cooking or steaming, have been reported to reduce the amount of tannin and its harmful side effects (Frutos et al. 2004). Now a day, bioprocessing has vast applies in livestock and poultry feed, using the enzymatic capabilities of microorganisms. Furthermore, Lactobacillus plantarum is a safe microorganism that has the potential for high enzymes to break down and eliminate tannin compounds (Curiel et al. 2009). This bacterium has vast applications for food processing in the industries (Siezen et al. 2011). Material and

The Experiment was done in a completely randomized design with 7 treatments and 3 replicates. Experimental groups were treatments with or without bacteria, aerobic or anaerobic condition and 5 or 10 days processing periods. The moisture content of all samples was adjusted to 50% by sterile water. For each gram of experimental sample, 107 CFU of Lactobacillus plantarum bacteria were added and then, placed into the incubator at 37 ° C. In this experiment, phenolic and tannin compounds, pH, soluble carbohydrate and volatile fatty acids (acetic acid, propionic acid and butyric acid) were measured. Phenolic and tannin compounds were measured based on Makkar (2000) method. Soluble carbohydrate was measured by spectrophotometric method with standard raffinose solution (Dubois 1956) and volatile fatty acids (acetic, propionic and butyric acid) were measured by gas chromatography. Soluble carbohydrate in four treatments (zero day and ten days without bacteria, ten days with bacteria in aerobic and anaerobic conditions) and volatile fatty acids in three treatments (10 days without bacteria and 10 days with bacteria in aerobic and anaerobic conditions) measured. These treatments were selected to interpret and justify the main events of fermentation processes. Data were analyzed by SAS software (2011). The comparison of the means was done using Duncan's multiple range test at P<0. 01.

The findings of the experiment indicate that the highest reductions in the content of total phenol, non-tannin phenol, total tannin, condensed tannin and hydrolyzable tannin (39, 24, 49, 49. 5 and 59 %, respectively, see table 1 and 2) were observed after 5 days of processing and there was no significant difference between 5 and 10 days of processing. It is assumed that significant reduction in phenol and tannin compounds was the consequence of Lactobacillus plantarum enzymatic activity. A research showed that Lactobacillus plantarum were reduced phenolic compounds of pomegranate juice (Mousavi et al. , 2013). Stopping the decline in phenolic compounds after the 5 days could result in lowering the pH to below 4 (Table 3) and cessation the biological activity of the Lactobacillus plantarum (Hunget 1957), or decreasing the efficiency of the enzyme tanase (tannin aceyl hydrolase). Also, there was no significant difference between the total phenol, non-tannin phenol (Table 1), total tannin, condensed tannin and hydrolyzable tannin percentage (Table 2) of aerobic and anaerobic treatments, which showed that the biological processes reduced phenolic compounds in aerobic or anaerobic conditions with the same efficiency. It has been reported that the enzymatic processes of breaking tannins compounds by bacteria in aerobic conditions are slightly different from the anaerobic conditions, resulting in different end products (Bhat et al. 1998). Our research findings show that the acidity of experimental samples in aerobic treatments was more than anaerobic in the 5 or 10 days of bioprocessing period (P<0. 01). Smetankova et al. (2012) showed that Lactobacillus plantarum grows faster in aerobic conditions and therefore, the pH decreases more quickly. After the processing, the amount of fatty acids (acetate, propionate and butyrate) increased, but the percentage of soluble carbohydrate decreased (P<0. 01). Table 4 shows that acorn kernel contains high percentage of soluble carbohydrate (on day zero), which is required for the growth and biological activity of Lactobacillus plantarum bacteria. Table 5 presents the results of statistical analysis of the treatments for the percentage of volatile fatty acids. The results showed that the percentage of acetic acid, propionic acid and butyric acid after 10 days of aerobic or anaerobic bacterial processing increased significantly, in comparison with non-bacterial treatment, but no significant difference was observed between aerobic and aerobic conditions, which indicates that in this study the activity of bacteria for the production of volatile fatty acids was not different in both aerobic and anaerobic conditions after ten days.

For the first time, this study has shown that in both aerobic and anaerobic conditions, the tannins compounds were broken down and reduced in equal proportion. The results of the experiment have shown that Lactobacillus plantarum bacteria can be used for bioprocessing of acorn kernel to reduce its tannins.

Appearance characteristics of the honeybee are important in identifying races and populations and because of their correlation with production and behavioral traits; they can be used to indirectly improve and reform these traits (Alpatov, 1929; Rinderer et al. , 1982). So, the first step in breeding of the honeybee is to perform morphological studies. In Iran, such studies are necessary for many reasons such as ecological conditions, topography, long beekeeping, and the climate of the four seasons. In 1927, the Russian researcher, Skorikov, identified an independent subtype called Apis mellifera meda, known as the Iranian honeybee, and introduced it to the world (Ruttner et al. , 1985). Tahmasebi (1998) also separated the entire Iranian honeybee population into three subpopulations of the north, west and northwest and central regions. Material and This research was carried out in 2 apiaries of 4 different townships in Zanjan province including Zanjan, Abhar, Mahneshan and Tarom. Identified colonies (40 colonies) include 5 colonies from 8 apiaries, and from each colony 50 (and a total of 2000) Apis mellifera young worker honeybees were collected, in which 800 bees (20 bees per colony) were evaluated. In this experiment, 17 apparent characteristics of the worker bees were evaluated included: the length of the proboscis, the length and width of the front wing, the length of the (a) cubital and (b) cubital veins, the number of right and left wing hooks, the discoidal shift, the length of the femur, tibia and tarsus, over hair, the width of third and fourth Tergitelongitudinal, cubital index, rear length, and the total length of the third and fourth ventral dentures. Measurement of morphological traits was done by routhener method and with the stereomicroscope equipped with eye graded lens. (Goetz, 1959; Dupraw, 1965; Ruttner, 1988). To analyze the variance of the data from the evaluation of traits, eight apiaries as treatments and five colonies of each were considered as repetitions. Analysis of variance was performed in a completely randomized design using MSTATC software. Then, the mean of traits in apiaries was compared with Duncan test. The apiary 1 of Zanjan with the more width of the third and fourth Tergitelongitudinal had the bees larger than bees of the other studied area and bees of the apiary 2 of Zanjan and apiary 1 of Abhar with the less width of third and fourth Tergitelongitudinal were smaller in body size than other bees. The observed difference in the size of bees in the apiary 1 and 2 of Zanjan may be due to the small size of the sample size in this study. The number of right and left wing hooks in the studied apiaries did not show any significant difference. The front wing of most (73%) of the studied bees in terms of the Discoidal shift was in the +1 position. The Discoidal shift feature was studied only by Farshineh Adl (2005) on Apis mellifera bees. The researcher concluded that 82. 5% of the apex wings of Apis mellifera were in the +1 position. Cluster analysis for all morphological characteristics divided the apiaries into two groups: (1) apiary 1 of Mahneshan, apiary 2 of Zanjan and apiary 1 and 2 of Tarom, (2) apiary 2 of Mahneshan, apiary 1 of Zanjan and apiary 1 and 2 of Abhar. In the principal component analysis for the 14 traits, the first and second components justified 50. 5% and 22. 8%, respectively, and in total 73. 3% of the total variation. The first principal component had the largest share in explaining the total variance. Width of wing, (a) cubital and (b) cubital veins, length of thigh, leg, tarsus, T3 and T4 and  left and right hooks were further explained by this component. Therefore, the above-mentioned traits, which are justified by this component, are more important than other characteristics in the distinction between bees in evaluated apiaries. The waist length, cubital index, and the length of the fifth dorsal abdominal hair showed the higher coefficient of determination in the second principal component. In other words, this component largely explains the traits that are of secondary importance. The twodimensional diagram of these two components was drawn, and the apiaries were grouped accordingly. The results confirmed the grouping cluster analysis. The studied apiaries were divided into two groups in terms of the 14 evaluated morphological traits. Width the wing, (a) cubital and (b) cubital veins, length of thigh, leg, tarsus, T3 and T4 and left and right hooks were the most important traits in the differentiation of evaluated honeybees.

Considering water and land limitation in agriculture, providing some year-round feedstuffs for livestock animals is a challenge for producers. Agro-industrial co-products, which are not in completion with human food resources, can be effectively consumed by ruminant species. Tomato waste (TW) is a by-product obtained after production of tomato sauce, paste and juices in manufactures. Using some physical and chemical processing of by-products can improve their nutritive value for feeding ruminants. Autoclave and exogenous fibrolytic enzymes are of those physical and chemical processing, which have been applied for several agro-industrial co-products. This study aimed to evaluate nutritive value, nutrient digestibilities and gas production parameters of chemically (autoclave and urea treating) and enzymatically (Natuzyme®) treated TW. Material and Fresh TW were sun-dried and were stored in cold temperature until the beginning of the experiments. Experimental treatments included: control (untreated), autoclaved (120 psi for 30 min), urea (3% urea added DM basis and anaerobically stored for 28 days in room temperature), and autoclave+urea (combination of previously mentioned treatments, 0. 5% enzyme and 1% enzyme). One day before the starting of experiment, aqueous solutions of enzymes were added to the samples. For the rest of experiments, samples were ground to pass 1 mm sieve size. Chemical composition of samples (i. e. , DM, NDF, ADF, CP and ash) was determined. For in vitro gas production tests (GP), the rumen fluid was taken from three ruminally fistulated Mehraban male mature lambs. For measuring kinetic parameters of gas production, 200 mg of samples were incubated in glass syringes containing 30 ml buffered-ruminal fluid for 144 hours. The cumulative produced gas was recorded at different times of incubation. The gas production data were fitted to exponential equation of GP= A (1-e-c(t-L) ); where GP is gas production at different times of incubation, A is asymptotic gas production (ml/200 mg DM), c is rate of gas production (/h), t is time of incubation and L is lag time (h). For measuring digestibility parameters, 500 mg of samples in glass syringes contained 40 ml buffered ruminal fluid were incubated at 39 °C for 24 hours. Volume of GP, metabolizable energy (ME), organic matter digestibility, partitioning factor (PF) and microbial biomass were determined after termination of incubation. All the in vitro gas production trials were carried out in three runs. Data were analyzed based on a completely randomized design using Proc GLM of SAS software. The differences among treatments were evaluated using Duncan's multiple range test. The highest amount of NDF, ADF and ash were observed in control treatment, while the highest CP was recorded with the treatment of 3% urea. The lower cell wall content in treated TW could be due to the role of urea and enzymes in breaking the links between cellulose and lignin. The treatment of 1% urea had the highest increment in comparison with control which is likely because of the activity of added enzymes in increasing fermentation of TW cell wall. The lower GP of 3% urea and autoclave+urea among all treatments can be due to the negative effect of ammonia on both direct GP from microorganisms activities and indirect GP from buffering of produced volatile fatty acids during fermentation. Also, the highest values of L were observed in 3% urea and autoclave+urea groups. The treatments were significantly different in dry matter  digestibility and OMD of which control had the lowest value. This could be due to the higher percentage of cell wall in control TW and the negative relation of cell wall with digestibility. The treatment of 1% enzyme and control showed the highest and lowest MB, respectively (P<0. 05). The higher MB in treated TW shows that processing had increased the availability of growth required factors for microorganisms. The PF were higher in urea, autoclave+urea and 1% enzyme groups (P<0. 05). The higher value for this parameter shows that degraded organic matter is more directed towards microbial biomass production rather than production of volatile fatty acids. The treatment of 1% enzyme had the highest value of ME, while control showed the lowest value (9. 57 vs. 9. 30 MJ/kg DM, respectively). Considering the obtained data related to dry matter digestibility, NDF, PF and chemical compositions of different treatments, it is concluded that the administration of 3% urea and using Natuzyme® at the level of 1% of DM improved the nutritive value of TW. Further research is needed to study the effect of using TW in performance of productive ruminants.

In this experiment the chemical composition, nitrogen fractions, degradation characteristics and gas production of raw and pelleted concentrate were determined. Food processing is one of the most important factors affecting feed intake and digestibility of feed which may be used to optimize fermentation and digestion (Dann et al. , 1999). Cereal processing can make optimal use of them, reduce nutrient losses, and also make livestock more balanced, which is very important in animal nutrition management. The aim of this study was to determine the effect of pelleting on the chemical composition, nitrogen fractions and degradability characteristics of commercial concentrates by two in vitro methods. Material and This study was carried out at the Research Farm, Faculty of Agriculture, University of Birjand, Iran. In order to prepare experimental treatments, concentrate samples were prepared in three stages. Step mash concentrates or Mash that was not inserted into pelleted machine, conditioner stage or phase that concentrates flour inside the machine under the influence of water vapor was 80-70 ° C and the third stage when the concentrates from the crossing conditioner cooled and turned into pellets. The treatments were: 1. mash concentrates before pelleting, 2. mash concentrates that has received heat and the pass conditioner and 3. Heated and pelleted concentrates. Degradability parameters of the samples were measured after incubation for 0, 2, 4, 8, 16, 24 and 48 hours in the rumen of two fistulated Holstein cows. The DM and CP degradation data were fitted by exponential equation P=a+b (1-e–ct). Effective degradability (ED) were calculated using of equation ED = a + { (cb) / (c + k) } and taking into consideration passing rate (k) 0. 02, 0. 05 and 0. 08 per hour. The gas production was recorded after 0, 2, 4, 6, 8, 12, 24 and 48 h of incubation. The data at the different times was analyzed using completely randomized design. The results showed the pellet process had no appreciable change on chemical composition (DM, CP, Fat, Ash, NDF, ADF) of concentrate. The non-protein nitrogen (NPN) decreased under the influence of heat processing. Neutral detergent insoluble nitrogen (NDIN) increased under the influence of heat processing (P<0. 05). Acid detergent insoluble nitrogen (ADIN) also increased under the influence of heat process but no significant difference observed. The lowest NPN was observed in heat-pelleted concentrate. Pellet heating caused a significant reduction in part b of dry matter and crude protein commercial concentrates. The rate of degradability in crude protein was associated with a significant reduction. There was a significant difference between the mean degradability coefficients and the effective degradation of crude protein under the influence of heat process. The highest mean slow part of degradation was observed in control treatment (0. 7360). Significant difference was observed between treatments in terms of fixed rate of degradability (P <0. 05), but there was no significant difference between two and three treatments. Pellet heating reduced part with the potential gas production that confirmed the results of the nylon bags method. According to the obtained data in this study, short chain fatty acid (SCFA), metabolizable energy (ME) and digestible organic matter (DOM) of experimental treatments showed significant differences (P<0. 05).

Conclusions: It was concluded that pellet heating reduces the dry matter and crude protein degradability, especially in both gas production and nylon bag technique. Therefore heat process during pelleting concentrate is a good strategy to reduce ruminal degradability of concentrate and supply bypass protein requirement of dairy cows.

Treatment of a dietary protein such as soybean meal (SBM) to decrease its solubility and thus, its hydrolysis to ammonia in rumen fluid should increase the amount of the dietary protein that bypasses ruminal degradation without sacrificing nitrogen required for ruminal microbial growth (Nishimuta et al. 1974). Protection of dietary protein from ruminal microbial degradation increases the supply of amino acids to the small intestine (Stern, 1981). Thus, the efficiency of protein utilization by the animal should be improved (Driedger and Hatfield 1972). Using suitable and practical processing methods for improving the nutritive value of feedstuff in ruminant nutrition have an important role in reducing production costs and improving production performance of ruminant animals. The aim of this study was to investigate the effects of various levels of tannin extracted from pistachio hulls on crude protein (CP) fractionation of soybean meal (SBM) and canola meal (CM) based on the Cornell Net Carbohydrate and Protein System (CNCPS). Material and Sun dried pistachio hulls were ground through a 0. 5 mm screen and soaked in water at a ratio of 1:10 (w/v) for 24 h. Then the pistachio extract was filtered and boiled to achieve pistachio extract concentrate (PEC) containing 11% total phenol and 7. 13% total tannin on a dry matter (DM) basis. The rates of PEC treatment were determined based on the previous in vitro findings of Dentinho et al. (2007), who found that treatment of SBM with crude extract from Cistus ladanifer L. caused a decrease in rumen degradation of SBM protein, even at relatively low phenolic doses (12. 5, 25 and 50 g total phenol per kg SBM). A total amount of 1 kg dried SBM and CM were ground through a 1 mm screen for each treatment. Both soybean meal and canola meal were treated with 0, 5, 10, 15, 20 and 30 % of PEC tannin. Finally, treated SBM and CM were airdried for 12 h to reach DM content of about 90% in whole product. The protein fractions of treated SBM and CM were determined according to CNCPS system. Analysis of phenolic compounds was conducted in three replicates as described by Makkar (2000). Total phenol was determined by Folin-Ciocalteu’s reagents, and the concentration was measured as tannic acid equivalent using tannic acid (Merck, Germany) as standard. Total tannins were measured as described by Makkar (2000). A completely randomized design with 6 treatments and 3 replicates was used for the study. The data were analyzed with general linear model procedure of SAS. Significance in the data was established when P<0. 05. Effects of SBM and CM treated with various amounts of PEC on chemical composition indicated that, adding PEC decreased DM, organic matter, neutral detergent fiber (NDF( and acid detergent fiber (ADF) content, but did not affect concentrations of CP for both SBM and CM proteins relative to its control groups. Vahdani et al. (2016) reported decreased NDF content of CM protein treated with tannin extracts from tea and pistachio. On the basis of the results, the lowest amount of soluble fraction (Sum of A and B1) was observed for CM at the level of 5% PEC tannin. There was no significant difference among treatments relative to control group in B2 fraction. Inclusion of PEC increased B3 fraction compared to control and 20% level has showed the highest B3 fraction. There was a reduction in C fraction by increasing level of PEC. The  lowest soluble fraction of CP followed by the highest escaped protein (B2+B3) elicited to recommend 5% level as the best treatment for CM, in lab scale. For SBM, the level of 30% PEC tannin increased bypass fractions of protein compared to other levels (P<0/05). This treatment changed the soluble (A and B1) (12. 37%) and insoluble fractions (B2, B3 and C) (87. 62 %) in SBM compared to the control and other levels of PEC supplementation. The results suggest that PEC supplementation decreases ruminal degradation of SBM, but increased rumen degradability of CM protein. There are some methods to decrease protein degradation in rumen such as using tannins. Tannins have been shown to decrease ruminal degradation of crude protein and increase the amount of CP that reaches the abomasum and small intestine (Alipour and Rouzbehan 2010). When using SBM treated with 10 to 250 g of quebracho tannins per kg, Frutos et al. (2000) also observed a decrease in in vitro intestinal digestibility of protein at the greatest treatment rate. Further research will be needed to determine if tannin treatment of dietary protein improves its digestibility. It has been demonstrated that feeding SBM-treated PEC increases average daily gain and feed efficiency in Holstein bulls (Jolazadeh et al. 2015). Overall, it seems that the tannin extracted from pistachio hull have different effect on CP degradability of SBM and CM. However, further research is necessary to investigate in vivo effects of these treatments

Trout (Oncorhynchus mykiss) is one of the most popular fish in the world. It is considered to as a best fish, and its pollution is important in terms of health and food hygiene. Trout is of the cold water fish and is in the subgroup of free fish. Animal protein, especially fish, has an important role in tissue retention, health and human happiness, and in general, the growth of the body due to the necessary amino acids and growth promoters, and the deficiency of this protein causes an imbalance in the nervous system, mental retardation, neuroticism, physical weakness in humans. and reduces the resistance of the body to infectious diseases. But, despite having a nutritional value and the body's need for aquatic animals, if the accumulation of heavy metals exceeds the limit, it can be a cause of disease and problem for humans, and therefore research in health matters is of particular importance. The presence of these compounds is harmful to human health and is not permitted. Heavy metals are environmental pollutants. The contamination of fish to heavy metals can lead to the accumulation of these elements in the body of consumers and the appearance of some diseases. Non-degradable pollutants such as heavy metal salts accumulate in the environment. The accumulation of toxic substances in the food chain may increase their concentration in animals at higher levels in the food chain. The accumulation and biological role of heavy metals in marine organisms can lead to the risk of human health from these metals. Exposure of fish to contaminants and its transmission to humans, can cause several diseases. The present research was aimed at determining concentrations of Lead and Cadmium, in muscles of farmed rainbow trout (Oncorhynchus mykiss) in Guilan’s Talesh, Iran, comparing levels of the metals in the flesh of both female and male rainbow trout, and comparing them with standards of Food and Agriculture Organization, Food and Drug Administration and Iran Veterinary Organization. Material and This research had 6 groups, consisting of trout which were 700-900 g and <300 g in weight. They were obtained in December, 2014. The homogenized flesh (15 numbers) was examined using an atomic absorption spectrometer. Trout fish was biometric. The trout were 478.56 g in weight and 318.93 cm in length. Despite pH and temperature, water hardness of studied pools showed significant differences (P<0.05). PH of water ponds was neutral. The water temperature was 15-16 ºC. The water temperature of the pool was not proper for heavy metals absorption to fish. Lead and Cadmium varied between 0.01> and 0.03. In this study, the amount of lead compared to Cadmium was not showed significant reduction(P<0.05). Hardness were 85.50 mg/l in A and B pols and 153.90 mg/l in pole C. Was not observed significant differences in water hardness factor between pools A and B compared with C pool. Water pools A and B were medium hardness and C pool was hard. Water of pools A and C were medium hardness, but water pool B was hard. Water hardness on cadmium in comparison to Lead had more effect. Correlation Lead and cadmium with age, length and weight in some of the pools were positive and in the others were negative. Between age groups similar were not observed significant differences in studied pools of Talesh (P>0.05). No significant differences in per farmed ponds between lead and cadmium concentrations in the age groups below 300 g 700 -900g(P>0.05). Except of the age group 300g< of pond A from Talesh, the other ponds in the age groups including of 300g< and 700 -900g was showed significant differences compared with FDA, FAO, WHO and Iran Veterinary organization in Lead and cadmium concentrations(P<0.05). There was no correlation between the accumulation of cadmium and lead compared with sex. According to achieved results and decreasing accumulation of heavy metals in farmed Trout Talesh compared with international standards, in terms of cadmium and lead contamination and food sanitary, farmed Trout of these regions are no side effects for human consumption in the autumn. According to the achieved results on farmed rainbow trout 700-900 g and <300 g in Talesh in fall, it must be declared that they cause no adverse effects and are fit for human consumption in terms of food hygiene and concentrations of lead and cadmium in them.

Introdution: A goal of ruminant microbiologists and nutritionists is to manipulate the ruminal microbial ecosystems to improve the efficiency of converting feed to animal products consumable by humans. The use of feed additives such as antibiotics has proven to be a useful tool to reduce energy (in the form of methane) and nitrogen (in the form of ammonia) losses from the diet. However, scientists have recently become interested in evaluating alternatives for manipulating the gastrointestinal microflora in livestock due to the increasing public concern about the use of antibiotics in the animal feed industry. Plant extracts have been used for centuries for various purposes (e.g., traditional medicine, industrial applications, food preservatives) due to their antimicrobial properties and because most of them are categorized as GRAS (Generally Recognized as Safe) for human consumption. The use of plant extracts appears to be one of the most natural alternatives to the antibiotic use in animal nutrition. Ruminant microbiologists and nutritionists have been exploring alternative methods of favorably altering ruminal metabolism to improve feed efficiency and animal productivity. Plant extracts contain secondary metabolites, such as essential oils (EO) that have antimicrobial properties that make them potential alternatives to antibiotics to manipulate micro-bial activity in the rumen. Essential oils are naturally occurring volatile components responsible for giving plants and spices their characteristic essence and color. Over the last few years, a number of studies have examined effects of EO, and their active components, on rumen microbial fermentation. Garlic oil (Allium sativa) and clove has been shown to exhibit a wide spectrum of antibacterial activity against gram-negative and grampositive bacteria. In a previous study, garlic oil and clove reduced the proportions of acetate and branchedchain VFA, and increased the proportions of propionate and butyrate and the small peptide plus amino acid N (SPep+AA N) concentration. These changes in the fermentation profile are consistent with those observed with methane inhibitors and have the potential to beneficially modify rumen microbial fermentation. In order to evaluate effects of garlic and clove essential oils on rumen microorganisms, dry matter degradability of ingrediends, two experiments were designed. Material and metheds: First experiment was carried out with three fistulated rams using change over design. Each period lasted 28d. Ruminal fluid was collected in the end of each period. Samplings were performed immediately before and after 2 and 4 hours of morning feeding. In second experiment: an experiment with randomized complete block design containing 3 kinds of feed (straw, alfa alfa, barley) and 2 kinds of essential oils (garlic, clove) in 3 replicates (3 fistulatted sheep) was conducted to determine effects of essential oil on degradability and effective degradability of dry matter. Incubation of feed samples was done in times of 0, 2, 4, 8, 16, 24, 48, 72 and 96 hours. The results showed that differences of pH were not significant among treatments. Clove essential oil did not show any effect on total counts of microorganisms, but garlic essential oil significantly reduced rumen total counts. Results of the experiment indicated that by using garlic essential oil in diet, numbers of producing lactic acid bacteria was significantly reduced but clove treatment didn’t affect numbers of producing lactic acid bacteria. Reduction in producing bacteria was observed in all the samples. Garlic and clove essential oil had not significant effect on the number of rumen coliform bacteria, but significantly decreased rumen protozoa bacteria. Number of rumen fluid protozoa was not significantly different from control before morning nutrition (time zero), but 4 and 8 hours after morning feeding, garlic and clove essential oil, significantly reduced rumen fluid protozoa. The results of this experiment showed that garlic and clove essential oil had significant difference with control in dry matter degradability, A, dry matter undegradibility and dry matter effective degradability by passage rate of 2%, 5%, 8% an hour. Significant difference was observed between straw, alfa alfa and barley in dry matter degradability, dry matter undegradibility and effective degradability. According to the results mentioned that garlic and clove essential oil effected on rumen microorganisms and dry matter degradibility. Plant-derived EO may be a useful means to improve efficiency of nutrient utilization in ruminants and reduce the impact of their production on the environment. At high doses, EO and their constituents may inhibit deamination of AA and reduce methane production in the rumen. The range of EO and their components is complex in terms of nature and activity and variability in their composition may make it difficult to obtain consistent positive responses in ruminant production from these complex mixtures

Recently, there has been increased research on isomers of conjugated linoleic acid. Conjugated linoleic acid (CLA) is a mixture of several geometrical and positional conjugated isomers of linoleic acid (LA, C18:2) which involve a double bond at positions 8 and 10, 9 and 11, 10 and 12 or 11 and 1, but the c-9, t-11 and t-10, c-12 isomers are physiologically important. CLA is produced in the rumen as a result of incomplete bio-hydrogenation of Linoleic acid. When bio-hydrogenation is incomplete, CLA can escape the rumen and be absorbed through the gastrointestinal tract, thereby providing the peripheral tissues with various isomers of CLA. Due to production of CLA isomers in ruminant’s rumen, ruminant products, such as milk and meat are main CLA sources in human diets (Chin et al., 1992). CLA has been reported to have anticarcinogenic, hypocholesterolemic, and antiartherogenic effects in laboratory animals. Some reports showed positive effects of CLA isomers on egg production, egg quality, immune response, antioxidant status and gene expression in laying hens. It has been documented that layer hens meet some oxidative attacks during force molting (Siegel 1980), therefore, the aim of this study was to evaluate the effect of different levels of conjugated linoleic acid (CLA) on egg production performance, some blood biochemical parameters and immune response of laying hens in post-molting phase. Material and In this experiment, sixty W-36 White leghorn laying hens in post-molting phase (78 week) were assigned to 3 treatments with 5 replications and 4 birds each based on completely randomized design. The purity of CLA source used in this study was above 90% with mixture of 50% c9-t11 and 50% c10-t12 isomers. Experimental diets were 1) Control diet (basal diet containing 0% CLA), 2) basal diet+0. 25% pure CLA and 3) basal diet+0.5% pure CLA and after adaptation period, diets were fed for 4 weeks. Egg production, egg weight, feed consumption, egg mass and feed conversion ratio were recorded weekly. In the end of experiment, two birds from each replicate close to cage average weight, was selected, blood samples were collected from the wing and serum separated. Total antioxidant capacity (TAC), total cholesterol, triglyceride and high density lipoprotein (HDL) cholesterol and low density lipoprotein (LDL) were measured using analytical kits. Another bird per replicate with average cage weight selected, after injection of vaccine in breast muscle, two weeks later, immune response was measured based on Hemagglutination-Inhibition (HI) Test. All data were analyzed by ANOVA using the procedure described by the SAS Institute (2009). Tukey test was used to determine the significant differences between treatment means. Results showed that egg production rate and egg mass of birds fed 0.5 % CLA were significantly higher than birds fed control diet or diet containing 0.25 % CLA during whole experimental period, but egg weight and feed consumption were not affected by dietary CLA level. Also feed conversion ratio of birds fed 0.5 % CLA was significantly (P<0.05) lower than control birds or those fed diet containing 0.25 % CLA. Ahn et al. (1991) reported improvement in egg production of layer hens fed 1.5 % CLA but Cherian et al. (2007) reported that egg production and egg mass of laying hens were unaffected by incorporating 0.25% CLA during the peak production period. Also Bolukbasi and Erhan (2004) reported no significant effect of 1% CLA in laying hens diet on the rate of egg production. Total cholesterol, HDL, LDL and VLDL in blood serum of birds were not affected by dietary CLA (P>0.05) in present study, but serum triglyceride decreased significantly with increase CLA level (P<0.05). Similarly, Munday et al. (1999) reported that mice fed CLA had lower serum triglyceride. The beneficial effects of CLA might be related to the fact that CLA decreases the activity of lipoprotein lipase (Park et al., 1997). Ha et al. (1990) reported that CLA is an effective antioxidant, more potent than a-tocopherol and almost as effective as butylated hydroxytolune (BHT), but total antioxidant capacity in blood serum of birds in present study were not affected by dietary CLA (P>0.05). It is concluded that use of 0.5% CLA in post-molting diet of laying hens improved immune system and egg production performance and decreased blood serum triglyceride of birds.

Adding genotypic and phenotypic information from cows to the reference population has the potential to increase the accuracy and decrease selection bias of genomic estimated breeding values (GEBV) in dairy cattle populations (Mc-Hugh et al. 2011; Thomasen et al. 2014; Wiggans et al. 2010). However, the cost of genotyping limits its use to an informative subset of animals (Boligon et al. 2012) and hence looking for an optimal strategy is crucial herein. Now a day, selective genotyping strategies (Boligon et al. 2012; Jiménez-Montero et al. 2012) and imputation methods (Calus et al. 2014) are some suggestive tools to reduce genotyping costs. Therefore, the aim of this study was to assess the effect of inclusion of cow genomic data in the reference population for genomic prediction under scenarios encompassing different selection strategies, number of genotyped cows and heritability on accuracy and bias of genomic breeding values when compared to the use of only bulls in the reference population. Material and A dairy cattle population was simulated by mimicking real linkage disequilibrium (LD) extent and population structure using QMSim software (Sargolzaei and Schenkel, 2009). This population mimicked a 4-pathway dairy cattle selection program with the use of artificial insemination (AI) technology, followed by a population with the use of genomic selection (GS). Within the GS reference population, pseudo-phenotypes were simulated for bulls and cows by calculating different predefined accuracies based on de-regressed EBV of Holstein cattle in Canada. That reflected difference between accuracy of EBV from a bull and accuracy of EBV from a cow which is based on his daughters’ records and her own and daughters’ records, respectively. Parameters of population simulation in this study were based on Dehnavi et al (2018). Two traits with a heritability of 0.30 and 0.05 (representing production and functional traits, respectively) were simulated independently. The reference population consisted of 5000 top selected bulls plus either 2500, 5000 or 10000 cows. Cows were selected randomly (R), based on the highest breeding values (H), the most accurate breeding values from top distribution tail (AH), two tails of the distribution of breeding values (TT), or the most accurate breeding values from two distribution tails (ATT) (Fig 1). The simulation was repeated 20 times for each scenario. Genomic predictions were computed using snpBLUP method in SNP1101 software (Sargolzaei, 2014). Pearson correlation, mean square error (MSE) between true breeding value (TBV) and direct genomic value (DGV), and coefficient regression of TBV on DGV were calculated as measurement criteria for accuracy, error and bias of genomic predictions, respectively. The extent of linkage disequilibrium in the simulated AI population showed a similar pattern that observed in North American Holstein cattle (Fig 2). The ascertainment bias was introduced according to the minor allele frequency (MAF) distribution observed in North American Holstein data (Fig 3). The ATT scenario resulted in higher accuracy of GEBV (Fig 4), lower MSE (Fig 5) and bias (Table 1) compared to other scenarios. This scenario led to 0.123 to 0.215 gain in accuracy (∆ accuracy) compared to the use of bulls only for traits with heritability equal to 0.30 and 0.05, respectively (Fig 4). R and TT scenarios followed ATT. Gains in accuracy were 0.117 to 0.204 and 0.113 to 0.196 for scenarios R and TT, respectively (Fig 4). Regardless of EBV accuracy, selecting cows with high breeding values (H, AH) led to the lowest accuracy of GEBV (Fig 4) and the highest MSE (Fig 5) and bias (Table 1). Overall the observed gains in accuracy for the two traits varied from 0.062 to 0.149 and 0.009 to 0.07 for scenarios AH and H, respectively (Fig 4). An increase in the number of cows decreased both MSE (Fig 5) and bias (Table 1). The average regression coefficient of TBV on DGV across all scenarios varied from 1.12 for 2500 cows to 1.08 for 10000 cows for the trait with a heritability of 0.30 (Table 1). These coefficients ranged from 1.16 to 1.14 for 2500 to 10000 cows for the trait with heritability of 0.05 (Table 1). Similar to these results, using Guernsey breed Jenko et al (2017) showed when only half of population was genotyped, genotyping cows with phenotypes in extremes was superior up to 8-10 folds in accuracy for yield traits than genotyping cows at random or genotyping cows with upper tail phenotypes. Genotyping cows with tail phenotypes can cover on average 88% of the difference between the scenario where all the cows were genotyped or only half of them were genotyped at random (Jenko et al. 2017). The present study showed that sampling cows from the most accurate EBVs on extremes seemed to be more informative on all SNPs, both favorite and unfavorite alleles in the population. The inclusion of cows in the reference population increased the accuracy of the genomic predictions across all scenarios and decreased bias of them as well. However, sampling cows from the most accurate EBVs from two distribution tails seemed to be more useful to precisely and accurately predict genomic breeding values of young animals than the other sampling strategies that were investigated in this study.

Estimates of the amount of milk produced by lactating ewes provide information for the implementation of optimum management and feeding strategies for ewes and their lambs. Lamb survival and subsequent birth weight gains until weaning reflect milk production abilities of ewes. Genetic evaluation of milk yield traits in dairy sheep may be based on whole lactation records (Rahman Ahmad, 2010) or test day records (Horstick et al. 2001; Komprej et al. 2009; Carlos 2014). The random regression method of data analysis seems to be the most appropriated technique to apply in longitudinal data including growth and milk production and the corresponding models have been incorporated in animal breeding for the estimation of breeding values (Schaeffer, 2004). The main objective of this study was genetic analysis of test day milk records for Kurdi ewes of Shirvan Kodri sheep breeding station using fixed and random regression models. Matherial and Kordi sheep is an Iranian fat-tailed native breed developed mainly for meat production but produce milk and wool production as well. This breed is distributed in North Khorasan Province of Iran and is mainly grown traditionally by nomadic people in pastures. Kordi sheep breeding station is located in Shirvan city, North Khorasan Province, Iran. This station was established in 1988. The breeding system in this station is semi-intensive. Considering local weather conditions, lambs and ewes are usually sent to the pastures around the station for daily grazing in late April. Sheep are grazed in the pasture until early July and during the wheat and barley harvest in farm lands of the town. With the onset of the cold season the herd is transferred to the station, and by setting appropriate diets each animal group (pregnant ewes, male and female lambs and rams) is fed separately and manually three meals a day. Mating season is from mid August to late October and the lambing begins in early January and continues until late March (Saghi et al, 2014). Data included 1124 test day records of milk yields collected from 250 Kurdi ewes. Milking was carried out by hand combined with lamb suckling at 14 days intervals starting from May to August 2012. General linear model was used to identify effective fixed effects on the trait by SAS 9.1 software. Fixed effects of litter size, parity and month of recording and random effects of direct genetic and permanent environmental effects were included in the models. Variance and covariance components were estimated using restricted maximum likelihood procedure. Genetic analysis of milk yield was carried out using fixed and random regression models by Wombat software. Average milk yield of studied Kurdi ewes was 0.38 kg. Average heritability estimates of milk yield were 0.04 and 0.07 in fixed and random regression models, respectively. In random regression model, the highest and the lowest heritability estimates were found at 14 (0.18) and 84 (0.006) days, respectively. Heritability of milk yield was higher in the first than in the second part of lactation period. Repeatability of milk yield in different days of lactation was ranged from 0.51 (day 28) to 0.89 (day 126). Genetic correlations between milk yields at different days in milk were ranged from -0.942 to 0.999. Permanent environmental correlations between milk yields at different days in milk were ranged from -0.850 to 0.920. Milk yield additive genetic and permanent environmental correlations between adjacent test days were more than between distant test days. Kominakis et al. (2001) were found low heritabilities for milk yields in different days of lactation for dairy ewes (0.05 to 0.25). Komprej et al. (2013) were also reported estimated heritabilities of milk yields in different days for Slovenian dairy sheep ranged from 0.08 to 0.16. Lower genetic correlations between milk yields at different days with increase in intervals between test day records were also found by other researchers (Zavadilova et al. 2005; Madad et al. 2013). Results indicated that Kordi sheep has a relatively good potential for milk production among Iranian native sheep breeds. For milk yield in the studied population, orthogonal Legendre polynomials of order 2 and 3 for additive genetic and permanent environmental effects, respectively, was found the most appropriate model. Considering low heritability of milk yield, improvements in environmental conditions could result in increasing milk yield performance in the studied flock. Lower genetic correlations with increase in intervals between test-day records may suggest that different genes are involved in milk yield in different days of lactation.

Medicinal herbs such myrtle leave are used in animals, and secondary metabolites of them alter rumen fermentation in livestock. Essential oils of myrtle leaf are containing terpinolene, cineole, linalool, terpineol and linalyl acetate. Also there are phenolic acids such as gallic acid, vanillic acid and ferulic acid; tannins such as galotannin and flavonoids such as myrcetin, catechin and quercetin in myrtle oil. Myrtle leave was used traditionally in the treatment of bronchitis, sinusitis, diarrhea and hemorrhoids. The studies showed that myrtle essential oils are used greatly as a food preservative. Also researchers reported that secondary metabolites of medicinal plants have hypoglycemic properties and reduce cholesterol and other blood lipids. There is rare information about using of myrtle leaf in livestock nutrition. Therefore, purpose of this experiment was to evaluate the effect feeding of myrtle leave on the gas production characteristics and fiber digestion and protein in Arabi sheep and effect of myrtle essential oils on gas production parameters. Material and In this trial, 8 Arabi sheep (about 9 months’ age and average weight 23±1.5 kg) were used and fed with diet without myrtle leave and diet containing 0.4 percent myrtle leave in diet 70:30 concentrate to forage for 30 days (4 replicates per treatment). In the end of experiment, fermentation parameters, gas production and in vitro digestibility of wheat straw and soybean meal with rumen fluid of sheep’s fed with experimental diets were measured. Rumen fluid was collected from animals before the morning feeding. About 200 mg sample (1.0 mm screen) incubated in 100 ml vials with 35 mL buffered rumen fluid under continuous CO2 reflux for 2, 4, 6, 8, 10, 12, 16, 24, 48, 72 and 96 h, in a water bath maintained at 39°C. Cumulative gas production data were fitted to the exponential equation Y=B (1−e−ct), where B is the gas production (mL) from the fermentable fraction, C is the rate constant of gas production (mL/h), t is the incubation time (h) and Y is the volume of gas produced at time t. Partitioning factor (PF), microbial biomass and truly digested organic matter was calculated by Makkar and Becker. For determination of partitioning factor at the end of each incubation period, the content of vials was transferred into an Erlenmeyer flask, mixed with 20 mL neutral detergent fiber solution, boiled for 1 hour, filtered, dried (in oven at 60 °C for 48 h) and ashed. In the second trial, the effect of different levels of myrtle essential oils (0, 0.05, 0.1 and 0.15 mL) in diet 30% forage and 70% concentrate on gas production parameters was measured by gas production method. Essential oils of myrtle leaf were taken by clevenger. Digestibility of dry matter and NDF of samples (wheat straw and soybean meal) were determined using tilly and terry method. Rumen fluid was collected from animals, and were mixed with McDougall buffer in a ratio 1:4. After gasifying with CO2, tubes were incubated at 39 ˚C. After 48 h of fermentation, 6 mL of 20% HCl solution and 5 mL pepsin solution were added and the incubated for 48 h, simulating post-ruminal degradation. After incubation, the residual substrates of each tube were filtered and used to determine digestibility of DM and NDF. The obtained data were analyzed in a completely randomized design using the General Linear Model (GLM) procedure of SAS software, version 9.1. The Duncan multiple range test was used to compare means at P< 0.05. Result and The results showed that no significant difference between treatments was observed in gas production potential of wheat straw, but gas production potential of soybean meal with rumen fluid of animals fed with myrtle leave after 96 hours’ incubation (158.13 mL/hour) increased (P<0.05). The gas production rate of wheat straw and soybean meal increased by myrtle leave (P<0.05). Fermentation characteristics (partitioning factor, truly digested organic matter and microbial biomass) of wheat straw were not affected by experimental treatments, but the partitioning factor, truly digested organic matter and microbial biomass of the soybean meal in animals fed with myrtle leave was higher in compared with control treatment (P<0.05). On the base of second experiment, using different levels of myrtle essential oils in diet significantly reduced gas production potential and rate and truly digested organic matter in comparison to the control treatment but PF significantly increased (P<0.05). Other parameters did not affect by levels of myrtle essential oils. It seems the inhibitory effect of essential oils on rumen microbial fermentation and decrease of digestion and gas production because of high content of active matters of essential oil in compared with myrtle leave, but increasing of gas production by inclusion of leave in diet refer nutrient availability of myrtle leave for microorganisms. The researchers reported cineole, alfa-pinen and tannins of myrtle essential oils caused to inhibition of microbial enzymes and decrease fermentation. Tannins can reduce microorganism adhesion to nutrients, inhibit microbial activity which have negative effects on fermentation and methane production. According to the result, it appears that inclusion 0.4 % myrtle leave in diet increased gas production and digestibility of NDF in Arabi sheep. But, myrtle essential oils levels significantly reduced gas production, fermentation and digested organic matter.

Corn meal is a by-product of corn grain which is a relatively new feedstuff in ruminant nutrition. This feed stuff is a by-product of corn after oil extraction. There is lack of information regarding the chemical analysis as well as its nutrient degradation in rumen. Moreover, the nutritional characteristics did not compare with other grains yet. Therefore, identification of its nutritional value is necessary for ration formulation. The aim of the present study was to evaluate and compare the nutritional value of corn meal with different corn varieties based on gas test technique as well as identification of microbial mass and short chain fatty acids (SCFA) concentration predicted based on this technique. The microbial protein production prediction was estimated as well to evaluate and compare the potential to produce microbial protein in corn meal and other grain sources. Material and Different grain sources which had been used extensively in local dairy farms had been used to compare their potential for gas production with corn meal as new feedstuff. The treatments were as follows 1) corn meal, 2) ground barley grain, 3) ground corn grain (contain semivitreous starch), 4) ground corn grain (contain floury starch), 5) ground corn grain (contain vitreous starch), 6) ground dent corn grain, 7) pure corn starch. Different grain sources were categorized based on starch form content. All the samples were ground to pass 1 mm sieve. The rumen liquor was collected from three non-lactating, non-pregnant dairy cows fed a similar basal diet. The gas production technique was conducted in three periods for and each sample has three subsamples in each period (totally nine repeat for each experimental feedstuff). Gas production was measured on 2, 4, 8, 12, 24, 36, 48, and 72 h after incubation. The measurement was done based on gas produced for fast and slow degradable fractions of feedstuffs and total volume was measured based on total gas produced through 72 h after incubation. In addition to gas production measurement, other fermentation parameters such as partitioning factor (PF), organic matter digestibility (OMD), microbial biomass production (MBP), metabolisable energy (ME), SCFA were estimated as well. In addition, the cumulative gas produced based on ml gas produced per mg of dry matter for different experimental feedstuffs was calculated. Results showed that corn meal has the lowest gas production volume from rapidly degradable pool, and floury starch contained corn had the highest rate of gas production volume. Slowly degradable pool of corn meal, also, has the highest (greatest) gas production volume. Despite these differences among treatments regarding the gas production in different sampled times, however, the total amount of gas produced in the different treatments was not significant. The results show that the proportional ratio of gas production was different based on different incubation times, but total produced gas was similar among treatments. The results show that cumulative gas produced (ml/mg DM) was differed between treatment with the lowest value for corn meal (124.48 ml/mg DM). The prediction results based on gas test method showed that digestibility of dry matter and organic matter from corn meal was lower in comparison with all varieties of grain corn. This may have related to chemical composition of this feedstuff in comparison to other treatments. Partitioning factor was statistically constant among treatments. Microbial biomass production as well as its efficiency was similar among treatments. This shows that corn meal was as same as other grain sources in affecting the microbial fermentation in vitro. Furthermore, metabolizable energy and predicted volatile fatty acids concentration of corn meal had the lowest value among treatments (10.30 Mj/Kg DM). The lower concentration for volatile fatty acid (1.10 mmol) predicted for corn meal clear that this by-product could not supply as adequate energy as supplied by other treatments for animal on farm scale. The lower ME content as well as the lowest SCFA predicted in corn meal treatment may be related to greater fiber content in comparison with other treatments. The greater cell wall content has potential to reduce energy content and total volatile acid production in rumen. Although the predicted SCFA concentration of other grain sources were greater than that of corn meal, all the predicted values were similar among grain sources. This was mostly because similar chemical analysis among these grains. The results of the present study showed corn meal had lower energy content compared to corn grain varieties as well as barley grain. However total gas volume production and microbial biomass produced for this feedstuff was similar to other experimental feedstuffs. Future studies need to evaluate the performance of the animal fed with corn meal when compared with other conventional grains.

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.

Identifying of genes with large effects on economically important traits, has been one of the important goals in sheep breeding. Over the last decade, by the advent of genome-wide panels of single nucleotide polymorphisms (SNPs), it has become possible to identify and localize QTLs for complex traits in many livestock species. One important obstacle in association studies is the confounding effect of population structure. Because this effect generally increases in proportion to population size, population structure remains a major concern in association analyses. To date, 98 QTLs for wool traits have been reported via genome scan based on marker-QTL linkage analyses (http://cn.animalgenome.org/cgi-bin/QTLdb/OA/index, 27 Aug, 2017). Compared with traditional QTL mapping strategies, a GWAS has major advantages both in its power to detect causal variants with modest effects and in defining narrower genomic regions harboring causal variants for economically important traits. In this study, we assessed population stratification and performed a genome-wide association study (GWAS) of wool quality traits in Zandi sheep. Material and

A total of 96 Iranian Zandi sheep was used in the study. The following four traits were analyzed: staple length (SL), mean fiber diameter (MFD), fiber diameter coefficient of variation (FDCV), and the proportion of fiber that was equal or more than 30 µm (F≥30). Animals were genotyped using 50 K SNPChip panel. Quality control of the genotype data consisted in removing SNPs with a call rate less than 95 %, SNPs with a minor allele frequency (MAF) less than 5 %, SNPs with more than x % missing genotypes, and SNPs that deviated strongly from Hardy– Weinberg equilibrium (P < 10−6). The PLINK was used to generate the matrix using the GLM algorithm. In this study, although the resources of this breed were very clear, we still examined the distribution of the test statistics obtained from the numerous association tests. We also assessed their deviation from the expected distribution of no SNPs being associated with the trait of interest using a quantile-quantile (Q-Q) plot, which is commonly used to analyze population stratification in GWAS. We use SNPEVG tool to show the (Q-Q) plot. The Bonferroni method was used to adjust for multiple testing from the number of SNP loci detected. We declared a significant SNP at the genomewide significance level if the raw P-value was0.05/N, here N is the number of SNP loci tested in the analysis. The exact positions of the annotated genes were extracted from the latest sheep genome Oar_v4.0 assembly along with the NCBI annotation release 102 of the sheep genome. To investigate if the significant SNPs detected in this study were within the range of previously identified QTL for relevant traits, we searched for meat or production QTL in the Animal QTLdb within a 1-Mb region on both sides of each significant haplotype.

After quality control, 2 individuals were excluded, leaving 94 sheep for the association analysis. Additionally, we removed 1070 SNPs with call rates less than 95% and 7717 SNPs with MAF less than 0.05. A total of 40,879 SNPs passed these quality-control filters and were retained in the dataset. These SNPs were distributed across 26 autosomes, with the number of SNPs per chromosome ranging from 747 to 5694, and with a mean distance between adjacent SNPs ranging from 50.4 to 68.7 kb. Wool MFD was 29.85±0.03 µm with an individual sheep range of 22.4-39.04 µm. The overall coefficient of variation of fiber diameter was 43.12%±0.7% with an individual sheep range of 19.7%–68.0%. The average percentages of fiber that had equal or more than 30 µm were 27.04±0.03% with an individual sheep range of 12.04-43.10%. Average wool staple length was 11.25±0.03 cm with an individual sheep range of 6-19 cm. The result from genomic control showed weak population stratification for SL, MDF, FDCV and F≥30 between populations of Zandi sheep. The genomic inflation factors (λgc) for the four traits were equal to 1.127, 1.101, 1.059, and 1.009 for MDF, FDCV, FD≥30 and SL, respectively. However, the Q-Q plots clearly showed there was no evidence of any systematic bias due to population structure or analytical approach in our case. Overally, two significant SNPs at the genome-wise level were identified for FD, and FDCV. No significant SNPs was identified for SLor FD≥30. Two Haplotype region within ERBB2 and GNAS genes previously reported in human growth and development hair and skin. Haplotypes were located with previously QTL reported to affect fiber diameter and fiber diameter coefficient of variation in Merino and INRA401 breed sheep. The functions of all of the above genes are directly or indirectly related to skin and hair development. Hair follicles are skin appendages and produce hair; therefore, we hypothesize that these genes control hair follicle development and fiber diameter trait.

The results of this study could provide a suite of novel SNP markers and candidate genes associated with wool traits and hence, may play an important role in understanding the biology of wool traits in fat-tailed sheep.