فصلنامه پژوهشهای علوم دامی ایران
سال شانزدهم شماره 2 (تابستان 1403)

The dry climate and water scarcity in most areas of Iran caused to serious restriction in planting fodder. Therefore, it is very important to find new fodder sources to feed ruminants in order to reduce the ration costs. One of the new sources of fodder that may have a favorable nutritional value are plants of the Paulowniaceae family in China, East Asia and some parts of Iran. Several types of Paulownia are known in the world. These plants are known for their high growth rate and intense photosynthesis, which are specific to C4 plants, and with wide leaves with a diameter of about 90 cm. The commercial production of Paulownia wood generates a large quantity of leaves, which are typically used as natural fertilizers.At the age of about 6 to 8 years, the production leaf of each Paulownia tree reaches about 100 kg. When the leaves have a favorable nutritional value, they can be used as fodder in feeding ruminants. In Iran, there has been no study on the nutritional value of Paulownia leaves for use in animal feed. Therefore, the aim of this research was to investigate the chemical composition of Paulownia leaves and the chemical composition, gas production parameters, digestion and fermentation characteristics of the silages from the mixture of different levels of Paulownia leaves (Tebjoo hybrid variety) and fodder corn in vitro. The corn forage and Paulownia leaves (Tebjoo hybrid variety) were prepared. At first, the chemical composition of corn forage and Paulonia leaf samples was determined based on the conventional methods. Then, both forages were chopped into 2-4 cm sizes using a hand chopper for silage preparation. Experimental treatments included silages containing different ratios of fodder corn and Paulownia leaves (0:100, 75:25, 50:50, 25:75 and 0:100). The materials were compacted in the plastic containers and the silage period lasted 60 days. Determining the chemical composition of fresh fodders and the nutritional value of experimental silages including chemical composition, in vitro gas production and fermentation parameters, digestibility and enzyme activity were carried out based on a completely random design. Results indicated that dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), ether extract (EE), lignin, non-fibrer carbohydrates (NFC) and water soluble carbohydrates content of fresh Paulownia leaves were 266, 911, 173, 402, 201, 35.5, 86.4, 404 and 91.2 g/kg DM, respectively. The mentioned chemical composition in Paulownia silage were 273, 902, 164, 393, 191, 38.8, 74.5, 386 and 25.4 g/kg DM, respectively. With increasing the proportion of Paulownia leaf in silage, DM, CP, lignin, EE, ammonia nitrogen concentration increased linearly (P<0.05). However, OM, NDF and ADF decreased linearly (P<0.05). By increasing the ratio of Paulownia leaf to corn in th silage, in vitro gas production (GP) in 24, 36 and 48 h, GP potential (coefficient b) and short-chain volatile fatty acids concentration decreased linearly (P<0.05), although it linearly increased ammonia nitrogen concentration and microbial protein synthesis (P<0.05). The carboxymethyl cellulase and filter paper degrading activities decreased linearly with increasing the inclusion level of Paulownia leaf in silage (P<0.05). However, ruminal alpha-amylase and protease activity showed a linear increase (P<0.05). The present study showed that the Paulownia leaf of Tebjoo hybrid variety has a favorable nutritional value as a new forage source in animal nutrition. Also, the preparation of silages including different ratio of Paulownia leaves and corn is recommended for animal feeding in the conditions of significant shortage of protein resources in the country. Future research is warranted to investigate how Paulownia leaf affect ruminant performance.

Although minerals include a small part of the total cost of poultry feed, they perform special functions in the body. Given that today's birds are primarily raised under stressful conditions (including high production demands, environmental stress, heat, and exposure to various other stress factors), their nutrient metabolism, including the absorption of minerals, has changed. This has led to decreased feed consumption, reduced mineral absorption, and consequently, increased excretion of minerals. Minerals that serve as cofactors for many enzymes are crucial; thus, to support proper functioning and enhance the immune system's response, higher amounts of these nutrients are required in diets. Chromium (Cr) is a critical micronutrient for humans and animals. It contributes to physiological and nutritional efficiency. Chromium increases glucose glycogenesis, promotes glucose transport, and increases protein synthesis. Chromium is important in protein digestion and reduction of lipid peroxidation and increased growth production. Chromium deficiency is associated with reduced growth rate, and glucose, and protein metabolism. Under environmental, nutritional, and hormonal stress, the benefits of chromium supplementation clearly demonstrated. Considering that with the increase in age and egg size, the egg quality decreases, and also the function of the immune system decreases at the late phase of egg production. Therefore, supplementation of layer hen diets with some nutrients at the end of the production period can improve the egg quality and potentiate the humoral immune response. Thus, the subject of the current study was to investigate the effects of dietary supplementation of chromium-methionine on egg laying performance, egg quality traits at different storage times and temperatures, humoral immune response, and some blood biochemical parameters of laying hens at late–phase of the egg production cycle.

A total of 120 Leghorn laying hens (Hy-line W36, 73 weeks- old) were assigned in a completely randomized design with 6 dietary treatments, 5 replicates and 4 laying hens in each replicate for 6 weeks. The experimental treatments were 6 levels of chromium-methionine supplementation (0, 400, 800, 1200, 1600, and 2000 μg/kg of diet) of corn-soybean meal-based diet. The hens were housed in a 60 × 60 × 40 cm cage. All hens were housed in an environmentally controlled house with temperature maintained at approximately 25ºC. The house had controlled ventilation and lighting (16L: 8D). All hens were supplied with feed and water ad libitum. The hen-day egg production, egg weight, egg mass, feed intake, and feed conversion ratio were recorded daily. The feed conversion ratio was expressed in kilograms of feed consumed per kilogram of the egg mass. The effects of organic chromium supplementation of laying hens on egg quality traits were evaluated every 2 weeks at 75, 77, and 79 weeks of age. The egg shape index was calculated by dividing egg length to egg width. At the end of the experiment, to measure certain blood parameters, one bird from each replicate was selected, and blood was drawn from the wing vein. After preparing the serum, the concentrations of triglycerides, cholesterol, total protein, albumin, globulin, uric acid, alanine transaminase, aspartate transaminase, glucose, and malondialdehyde were measured using Pars Azmoon kits according to the manufacturer's instructions.

Supplementing the diet with chromium-methionine had a significant effect on laying performance (including feed intake, feed conversion ratio, and egg production) and egg quality traits (such as eggshell weight, yolk color, yolk weight, albumen height, and Haugh unit). The results showed that birds fed 2000 µg/kg chromium-methionine had the lowest feed intake and the best feed conversion ratio and egg production percentage were observed with the addition of 1600 and 2000 g/kg organic chromium. By suppressing stress factors, chromium can increase the ovulation rate and increase the percentage of egg production. Addition of organic chromium except 2000 μg/kg improved albumen height and Haugh unit compared to the control group. The interaction effect between egg storage time and storage temperature on the yolk height and yolk percentage, albumen and yolk pH were significant. Carotenoids are responsible for the color of the yolk and they are a part of the fat-soluble compounds, so any factor that can improve the digestion and absorption of fats will probably lead to an increase in the color of the egg yolk. Haugh unit is a measure to determine the quality of egg white, which determines the quality of the egg. The higher HU means better egg quality (fresher, higher quality eggs have thicker whites). Blood serum cholesterol of chickens fed with 800 µg/kg of chromium-methionine decreased compared to the control group. The lowest serum concentration of malondialdehyde was observed using 1600 and 2000 µg/kg organic chromium. Addition of 1600 µg/kg of chromium-methionine increased total immunoglobulin production against sheep red blood cells. The effects of dietary chromium supplementation on improving immune responses can be attributed to its antioxidant activity. By enhancing antioxidant activity, chromium reduces the production of corticosterone, a potent suppressor of immune system function. This reduction in corticosterone levels prevents the decrease in lymphocyte production and proliferation, ultimately supporting the continued production of antibodies.

In total, dietary supplementation of 1600 μg/kg chromium-methionine can improve egg laying performance including feed conversion ratio, egg production, and egg mass, as well as egg quality traits such as egg shell weight and albumen height of laying hens at the late-phase of laying cycle. Supplementation of layer diets with 1600 μg/kg organic chromium improved the humoral immune response at the late-phase of egg production cycle by increasing the production of total immunoglobulin against sheep red blood cells.

Due to genetic selection, today's strains exhibit higher metabolic activity, resulting in increased heat production and reduced heat resistance. This can negatively impact their immune system and production performance (Havenstein et al. 1994).Thaxton et al. (1968) showed that high ambient temperature can have a negative effect on the immune responses of broiler chickens. In recent years, in order to improve the health of chicken meat consumers, the production of broiler chickens without the use of antibiotics has been proposed, and the ban on the use of antibiotics has been issued by the European Union in 1999 (Youssef et al., 2016). For this reason, today there is a tendency to use alternative sources. Algae and microalgae are of the natural substances whose beneficial effects have received much attention in recent years. Spirulina platensis (SP) is a filamentous blue-green microalgae (cyanobacteria) generally regarded as prebiotic and source of high quality protein, minerals, essential fatty acids, essential amino acids, pigments and phenolic acids. Many studies have shown that Spirulina has antioxidant, immunomodulatory, anti-inflammatory, antiviral, and antimicrobial activity in various experimental animals (Hajati and Zaghari 2019).

A total of 360 one-day old male broiler chickens (Ross 308 strain) were allocated to 6 treatments, 6 replicates and 10 birds each replicate in a completely randomized design. The experimental treatments included different levels of Spirulina platensis microalgae powder: 0 (Control), 0.25, 0.50, 0.75, and 1.0 percent Spirulina platensis powder, and one treatment included Vitamin E at the level of 150 g/ton feed. The basal diet was based on corn0soybean meal and all the diets were isocaloric and isonitrogenous and used for 42 days in diets. During the experiment, the feed consumption and weight gain of the chickens were measured weekly and the feed conversion ratio was calculated for each week and period.On 42 d of experiment, 2 birds from each replicate were randomly selected, humoral immune response was evaluated and then killed after weighing, carcass components and internal organs were weighed and their weight was calculated as a percentage of live weight. In order to evaluate the humoral immune system, the antibody titer produced against SRBC was measured using the hemagglutination method. In the end of experiment, the microbial population of the cecum was counted. All the data were analyzed in the form of a completely random design by SAS software and with the GLM procedure, and the comparison of means was done with Tukey's test.

Results showed that the use of different levels of SP had no significant effect on the amount of feed intake during the whole period (P<0.05), but in finisher period, using %1 SP could improve body weight gain and feed efficiency of the broilers compared to control group (P<0.05). Breast yield of the chickens was affected by the level of dietary Spirulina, and it improved at the levels of 0.5, 0.75, and 1.0 percent SP usage compared to control and vitamin E groups (P<0.01). The relative weight of abdominal fat decreased in the birds fed with SP at the levels of 0.75 and 1.0 percent compared to control group (P<0.01). There was no difference among total count, ceca Lactobacillus and E. Coli in the chickens of different experimental groups (P>0.05). The response of humoral immune system was higher in all four levels of SP and also vitamin E compared to control treatment. Chickens which consumed Spirulina at the level of 1 percent had the highest level of antibody titer. Mustafa et al. (2021) by achieving a growth performance similar to the present research, stated that the effect of SP on the intestinal microbial population, such as the increase of Lactobacillus and the decrease of Escherichia coli, has improved the performance of the birds under heat stress conditions. According to the reports on the effects of heat stress on digestive enzymes (Sahin et al., 2002) and the efforts of previous researchers to overcome the adverse effects of heat stress on digestive enzymes by using phytogenic compounds (Khosravinia et al., 2016), the researchers of the present study believe that the improvement of the mucous tissue and intestinal immune system and perhaps the increase in the secretion of digestive enzymes are the most important factors for improving the performance of birds in heat stress conditions.

Under heat stress condition, using 1 percent Spirulina platensis microalgae in broiler chickens’ diet could improve both humoral immune system and growth performance.

Phosphorus (P) and calcium (Ca) are macro-minerals for normal growth and development. Phosphorus plays a vital role in muscle function, enzyme activity, lipid metabolism, bone mineralization, maintaining osmotic and acid-base balance, energy metabolism, amino acid metabolism, and protein synthesis. In addition, in layers, P is required to replace tissue metabolites such as nucleotides and phospholipids, maintain skeletal integrity, and produce the egg. Also, Ca is essential for eggshell formation, coagulation, muscle, and nerve function. Bone is a rich source of P and Ca, which can be converted into bone ash (BA) as an organic source to supply P and Ca. Today, BA has been introduced as a suitable alternative for mineral sources of P and Ca in the poultry industry. Barshan et al reported in 2019 that the use of BA compared to dicalcium phosphate caused a significant increase in weight in broilers. The purpose of this study was to use bone ash as a dietary P source and to investigate the P bioavailability in it compared to monocalcium phosphate (MCP) as a standard source. 350 one-day-old broilers were used in a completely randomized design with 7 treatments and 5 repetitions. The dietary treatments were: 1) basic diet containing 0.11% of available P; 2, 4, 3) supplementing the basic diet with 0.05, 0.1, and 0.15% P from MCP source; 5, 6, 7) supplementing the basic diet with 0.05, 0.1, and 0.15% P from BA source. All experimental diets contained 0.78% Ca. At the end of the experiment, the P bioavailability in the BA was obtained based on the P consumed in relation to the daily weight gain and tibia bone ash by the slope-ratio method. Also, the investigated parameters such as digestibility and antibody titer against sheep red blood cells (SRBC) were analyzed in a factorial design. The results showed that based on the weight gain in the grower (10 to 25 days) and finisher (26 to 35 days) periods, the bioavailability of P in BA was 82.37% and 93.24%, respectively. Therefore, it can be concluded that with the increase in the consumption time of BA, its P bioavailability has also increased and it has been able to compensate for the lack of growth well and almost similar to monocalcium phosphate. Also, based on the percentage of ash and P of tibia bone, the P bioavailability was 92.86% and 96.39%, respectively.The factorial analysis of digestibility results also showed that by increasing the level of P in diet and using MCP, the percentage of dry matter digestibility increases significantly (P<0.05). Also, the percentage of P digestibility increased numerically with the increase in diet P and the use of BA. Also, factorial analysis of the data related to the production of antibody titer against sheep red blood cells, we observed that the only significant difference in the main effects was for the total antibody titer and IgG in the secondary response; So that the antibody titer produced in the secondary response was significantly higher in chickens fed with monocalcium phosphate than in chickens fed with bone ash (P<0.05). The results of the bone quality test showed that the shear and tensile stress for the tibia was significantly higher in chickens fed with 0.26% P (P<0.05); but there was no significant difference between chickens fed with MCP and BA. An increase in tensile and shear stress means that more force is required to break these bones, which indicates the greater strength of these bones. Measuring the percentage of ash and phosphorus (P) in tibia bones showed that as the dietary phosphorus level increased, the percentage of ash and phosphorus in the tibia also increased significantly (P<0.05). Additionally, using monocalcium phosphate (MCP) in the diet resulted in a significant increase in tibia phosphorus (P<0.05); however, there was no significant difference in the percentage of tibia bone ash. Given the limited availability and high cost of phosphorus and calcium mineral resources, it is essential to find suitable alternatives. Bone ash (BA) is a rich source of phosphorus and calcium, which can be used as an organic source to supply these minerals in industrial poultry diets. Consistent with our results, Van Haren et al. reported that BA can compensate for growth deficits in broiler chickens, similar to diets supplemented with dicalcium phosphate (DCP) and MCP, compared to diets without a phosphorus source. In general, according to the results, it can be concluded that the bioavailability of P in BA is 92 to 96% compared to MCP. This result shows the good ability of BA to replace mineral sources of P. Also, since the absorbability of MCP is higher than dicalcium phosphate (DCP), it can be concluded that BA can have the same efficiency as DCP and be introduced as a substitute in the poultry industry.

Antibiotics have been used for many years in animal production to treat infectious diseases and as growth promoters. However, the misuse of antibiotics causes problems such as bacterial antibiotic resistance. Also, the possible accumulation of antibiotic residues in livestock products is risky for consumers. After the ban on the use of antibiotic growth promoters in 2006, there was a need to use substitutes in animal feed to replace antibiotics. These alternatives include prebiotics, probiotics, phytobiotics and synbiotics. Probiotics are live microorganisms that, when consumed in sufficient amounts, have beneficial effects on the host by creating a microbial balance in the gut. The purpose of probiotics is to create a competition between the species that are naturally present in the intestinal flora of broilers. The most important advantage of probiotics is that they do not remain in animal products. Prebiotics are feed compounds that are not digested by the host when consumed but can support beneficial bacteria. These compounds are short-chain carbohydrates such as non-digestible oligosaccharides that cannot be digested by animal enzymes. Compounds containing prebiotics and probiotics used in nutrition are called synbiotics. Phytobiotics include a wide range of plant-derived products with bioactive compounds. Plant feed additives (PFAs) are widely effective in improving gut health, increasing digestibility and thus growth performance. These bioactive compounds include secondary metabolites (phenolic and flavonoid). In recent years, various marine organisms have been considered as valuable biological compounds for livestock. Marine algae, due to a wide range of bioactive components such as flavonoid, carotenoid, phenolic compounds, tocopherol, peptide and various sulfated and carboxylated polysaccharides such as alginate and fucoidan with antibacterial, antifungal and antiviruses have beneficial effects on health. Brown macroalgae such as Sargassum angustifolium have beneficial effects on health due to a wide range of bioactive components such as fucoidan, fucose sulfate and polysaccharides. Also, this group of algae is a rich source of sodium alginate oligosaccharides.

The present study was conducted in the form of a completely randomized design, lasted for 42 days at the Ali Abad Kamin research farm of the Fars Agricultural and Natural Resources Research and Education Center, Shiraz, Iran. A total of 240 Ross 308 broilers were distributed in five treatments, four replications and 12 birds per replication. The dietary treatments included: 1 basal diet, without additives, 2 basal diet + 0.05% Oxytetracycline, 3 basal diet + 0.2% brown algae Sargassum and treatments 4 and 5 the basal diet was + 1% and 1.5% of synbiotics Limax, respectively. Ingredients and chemical composition of the ration are presented in (Table 1). At the end of each period, chickens in each group were weighed and the average body weight gain (g/b) in each period was calculated. Feed intake in each period was calculated and expressed as g/b/d. Based on weight gain and feed intake in each period, the FCR values of each group were calculated. Tow representative chickens from each group were selected for carcass analysis representing the average weight and variability of each group. The data obtained on various parameters studied during this experimental trial were analyzed statistically with SAS software.

The results showed that the treatments had no significant effect on the feed conversion ratio of the birds. In the finisher period (25-42 days) and total period (1-42 days), oxytetracycline and Limax 1% treatments had the highest and lowest average daily weight gain, respectively. There wasn't any significant difference between average daily weight gain of the oxytetracycline treatment and other experimental treatments. In the finisher period, the highest amount of feed intake was for the control treatment and the lowest for the Limax 1% treatment (P<0.05). The highest value of production index was belonged to oxytetracycline and Limax treatments was 1.5% (295 and 272, respectively). The highest abdominal fat percentage in the day 42 was related to Oxytetracycline treatment and the lowest was related to Limax treatment of 1.5% (P<0.05). The highest villus width in the day 42 was belonged to Limax treatment of 1 and 1.5% and the lowest was belonged to Oxytetracycline, control and Sargassum treatments (P<0.05). The treatments did not show a significant difference in the feed conversion ratio. The highest value of production index was observed in oxytetracycline and Limex 1.5% treatments (295.25 and 271.71, respectively).

The final result is that the two oxytetracycline and 1.5% of synbiotics Limax treatments shown the highest average daily weight gain and the production index among the experimental treatments. Also, the maximum width of villi and its absorption surface of villus was related to Limax treatment of 1.5%. Therefore, despite the problems of using antibiotics in the diet of birds, Limax synbiotic supplement can be a suitable substitute for oxytetracycline antibiotic.

Ascites syndrome, as a metabolic disorder, is one of the most non-infectious causes of death in the old age of breeding in the poultry industry, which causes annual losses of over one hundred million dollars and is an important economic concern. The growth rate of broiler and their high need for oxygen causes an increase in pumping, followed by heart failure, and heart failure is often associated with other diseases. Due to the relationship between the heart and the kidney, chronic heart failure leads to a decrease in filtered blood volume and a decrease in kidney function, which over time causes permanent damage to the kidneys. lncRNAs play important roles in a variety of different mechanisms related to cellular homeostasis and in a wide range of pathophysiological processes and pathogenesis of many diseases, including cardiovascular disorders, respiratory and kidney diseases, preliminary studies in human samples show shows that lncRNAs are strongly involved in the development and disease of the kidney. For this reason, the lncRNAs obtained from the RNA-seq data of the kidney tissue were investigated in the occurrence of ascites.

700 one-day-old chicks from one of the paternal lines of a commercial strain were kept under standard conditions until 21 days old. On the 21st day of the breeding period, cold stress (24 degrees Celsius) began and continued until the age of 48 days. After applying cold stress, the birds with ascites symptoms were grouped in the Ascites group and the rest in the Healthy group. On the 39th day of the breeding period, 70 slaughtered birds and kidney tissue samples from each bird were immediately transferred to the liquid nitrogen tank after separation. 16 ascites birds and 16 healthy birds were used for RNA extraction. Total RNA extraction was done individually using trizol (YTzol) solution, and then an equal amount of RNA from four tissue samples was combined and then cDNA was prepared from 4 ascites tissue samples and 4 healthy tissue samples. All small RNAs, such as rRNAs, tRNAs, etc., were removed by dt oligo beads, and finally, all mRNAs were used to prepare the library. Novagen company was used for sequencing using Illumine hiseq 2500 technology and paired-end reads. Several software such as hisat2, cufflinks, stringtie, cuffmerge and cuffdiff were used for mapping, aligning and analysis of gene expression differences. In order to extract coding and non-coding genes, FEELnc software was used with default settings. Potential target genes of lncRNAs were investigated by searching for protein coding genes located within 100 kb upstream and downstream of each lncRNA. In the following, a positive and negative correlation of 90% was obtained between two groups of coding and non-coding genes based on the degree of expression change. In order to investigate the metabolic, structural and functional pathways of significant genes, the Enrichr database, which is connected with other databases such as KEGG, was used.

In the current research, a total of eight samples produced 187640642 million paired reads with a size of 150 nucleotides and after quality control, 185258819 uncontaminated reads were obtained. The number of 1421 lncRNA transcripts related to 921 gene loci and 154 related target genes were identified in the comparison between ascites and healthy birds. Then, this number of genes were identified (154 genes) in order to check their functional characteristics using the Enrichr database, five functional pathways Glycine, serine and threonine metabolism, Rap1 signaling pathway, Sphingolipid metabolism, Phosphatidylinositolsignaling system, Mucin type O-glycan biosynthesis and related genes were significant. Out of 13 significant lncRNAs in these biological pathways, 12 are located on the antisense direction and one is located on the sense direction. In addition, 9 lncRNAs are exonic, 3 intronic and 1 in downstream position. These pathways are activated as damage modifiers in hypoxic conditions caused by ascites and provide the required energy and maintain kidney homeostasis in response to oxygen tension caused by ascites. On the other hand, they act as cell survival, increase proliferation and anti-apoptosis, which on the one hand reduce kidney damage and on the other hand make it function better, and in this way, reduce complications caused by ascites kidney damage.

Therefore, by targeting the important pathways of biology obtained and the genes affecting them for the prevention and treatment of ascites disease, it will provide a new insight for breeding.

Genetic architecture of sheep reproduction is increasingly gaining scientific interest due to the major impact on sheep production systems. The number of lambs per lambing is one of the most important reproductive traits in sheep. Many studies have reported that genetic mechanisms play an important role in the variation of litter size in sheep. Reproductive traits normally show low heritability and therefore response to conventional selection methods is not satisfactory for these traits. Considering the genetic information of the genetic variants underlying reproduction variability could efficiently increase the selection efficacy. Genome-wide association studies (GWAS) have been used to identify associations between genotypes and phenotypes as well as candidate genes for reproductive economically important traits. Statistical power in GWAS is mostly affected by sample size. The low sample size is hence a main obstacle in GWAS. Combining multiple data sets of different studies for joint (mega) GWAS provides an opportunity to increase the sample size required for GWAS. This study was performed to identify genomic regions affecting litter size in different sheep breeds using the mega-analysis of GWAS. Multi-population joint GWAS was performed using genotypic and phenotypic data of three sheep breeds including native Zandi and two breed retrieved from the database. Quality control was performed using the Plink software. The markers or individuals were removed from the further study based on the following criteria: (1) unknown chromosomal or physical location, call rate <0.90, missing genotype frequency >0.05, minor allele frequency (MAF) < 0.05, and a Pvalue for Hardy–Weinberg equilibrium test less than 10-6. Before analysis, imputation of missing genotypes for combined data set was implemented by LD-kNNi method. Mega-analysis was performed using a mixed linear model in TASSEL software considering kinship and population structure (top five components of principal component analysis (PCA)) as confounding effects. The quantile–quantile (Q–Q) plot was visualized by plotting the distribution of obtained vs. expected log10 (P-value). The association results along the genome and the significant SNPs were visualized in the Manhattan plot. To account for multiple test problem and identify the genome-wide significance level, Bonferroni test was used based on the number of independent SNPs obtained from pairwise linkage disequilibrium analysis. After GWAS analysis, the 500 bp sequence upstream and downstream of the significant SNP was explored to identify the adjacent candidate genes using ARS-UI_Ramb_v2.0 (Genome Data Viewer). In the present study, we implemented a mega GWAS using three different sheep breed data to identify the genetic mechanisms responsible for litter size in sheep. After quality control, 671 animals and 45167 SNP markers were kept for further analysis. The results of the mega-analysis identified nine marker on chromosome on chromosomes 1 (two SNP), 2, 3 (two SNP), 10, 13 (two SNP), and 22. The quantile–quantile plot that features the total distribution of the observed P-values (−log10 P-values) of quality passed SNPs vs. the expected values, showed the effective control for confounding effects. Many of the significant SNPs identified in this study were located in or very adjacent to known genes (DLG1, CLSTN2, INHBE, TCFL5, and RBP4) that have been already reported for their contribution to fertility and pregnancy success. It has been reported that the RBP4 gene is expressed during the period of fast elongation of the pig blastocyst which is a crucial period for the survival of the embryos. Also, it has been suggested that CLSTN2 has the main contribution in uterine and conceptus physiology during the establishment of pregnancy and therefore can be considered as a candidate gene for litter size. INHBE has an essential function during ovulation and pregnancy through extracellular matrix (ECM) components degradation and therefore enabling cell migration and angiogenesis. Comparison of the results of this study with previous reports showed that the mega-analysis of GWAS, compared to the meta-analysis already reported for GWAS results, had comparable power in identifying genomic regions influencing litter size in sheep but identified fewer genomic regions than individual GWAS for each breed. No previously reported major genes controlling litter size in sheep were identified using our mega GWAS. The results of our research are suggested for further investigations in identifying causal genetic variants or genomic regions underlying the litter size variation in sheep and can be used to understand the genetic mechanism controlling this trait.

So far, many researches have been conducted to compare the productivity of different silkworm hybrids in Iran, which were just based on the evaluation of cocoon traits, eg. cocoon weight, cocoon shell weight, cocoon shell percentage, pupation rate, etc. In the present study, with the aim of obtaining information on the quality characteristics of silk thread in Iranian silkworm hybrids, 80 Iranian silkworm hybrids were examined for silk thread characteristics. 72 of the new silkworm hybrids in addition to 8 current commercial hybrids were evaluated for the six silk thread characteristics including yarn size (YS), yarn length (YL), yarn tensile strength (YTS), elongation percentage (EP), yarn weight (YW) and raw silk percentage (RSP) based on dry weight. 200 good cocoons from each hybrid were sampled at the Iran Silk Research Center and sent to the Fiber Physics Laboratory of the University of Guilan. After re-evaluating the individual cocoons based on form and size of them, 3 repetitions of 21 cocoons (based on the capacity of the spinning machine) were spun and dried. According to reciprocal crosses in this research, the data were analyzed using a completely randomized design (CRD) with two sources of variation (40 hybrids and two types of crosses) by the GLM procedure of SAS software version 4.9. Mean comparison of the studied traits based on the combined reciprocal data (40 hybrids) showed that the highest average of the FS trait (73.3 deniers) was for the hybrid IRA3×IRA10 and the lowest average (58.2 deniers) for the commercial hybrid 153 ×154. The highest YL trait(771.4 m) was for hybrid IRA5×IRA8 and the lowest (441.18 m) for the commercial hybrid IRA2×IRA3. The highest average of the YTS (3.47 g/denier) was for commercial hybrid 104×103 and the lowest average (2.9 g/denier) was for hybrid IRA2×IRA3. The highest average for the EP trait (17.6%) was reported for the commercial hybrid 154×151 and the lowest average (12.83) was related to the hybrid IRA2×IRA3. The highest average for YW (5.59 grams) belonged to the IRA5×IRA8 hybrid and the lowest average (3.34 grams) belonged to the IRA2×IRA3 hybrid. Also, for the RSP trait (the most critical trait of silk characteristics), The IRA5×IRA8 hybrid had the highest average (41.4%) and the IRA2×IRA3 hybrid had the lowest average (25.86%). IRA5×IRA8 hybrid had superior performance than all commercial hybrids. The RSP of the IRA8×IRA9 hybrid was 39.47%, which was superior to the three commercial hybrids. The hybrids that were excellent in at least three of the six characteristics were: all commercial hybrids and three new hybrids including IRA5×IRA8, IRA11×IRA12 and IRA8×IRA9. Analysis of the variance of the traits showed that the reciprocal-cross effect was not significant for the YS and YTS traits (P>0.05). Despite the close competition of new hybrids with current commercial hybrids, many characteristics of commercial hybrids were significantly higher than new hybrids. However, the new hybrids including IRA2×IRA9, IRA2×IRA11, IRA3×IRA4, and IRA4×IRA11, which have shown superiority in terms of cocoon traits, also performed favorably in terms of all yarn characteristics. However, two hybrids were among the superior hybrids in terms of cocoon production and cocoon traits, but the results of the present research showed that they are not superior in terms of yarn characteristics and raw silk yield and productivity, including IRA2×IRA3 and IRA4×IRA7. The IRA2×IRA3 hybrid has an unfavorable performance in terms of weight, strength, and raw silk percentage and cannot provide the interests of silk spinners. In the final selection of new hybrids based on the performance of both cocoons and silk thread, it is necessary to pay attention to the strength and weakness of the silk yarn obtained from the cocoons of each hybrid. But it will not be the case that hybrids with better cocoon performance are necessarily among the best in terms of all silk yarn traits or even some of them. For example, hybrids IRA2×IRA9, IRA2×IRA11, IRA3×IRA4, and IRA4×IRA11 had better cocoon performance, but they had a middle performance for yarn characteristics. In other words, if they are not among the weaker group, they can be considered. Generally, it is necessary to decide about new hybrids after rearing them in rural conditions and evaluating the produced cocoons and yarns.

Sheep are the main source of wool and its fiber characteristics, such as diameter, length, and color, which are determined by genetics and environmental factors, are key features in the economic value of sheep wool. In sheep, white wool has the highest economic value due to its dyeability, thus, the identification of mechanisms responsible for coating color determination is very important from an economic point of view. In general, the coat color is determined based on the amounts and types of melanin produced and released by the melanocytes in the skin tissue (Ito et al., 2000). The genetic basis and genes involved in coat color are well understood in rodents, although many of these genes are incorporated in coat color regulation in other species; including Sheep also have a common role. In Iran, the Lori Bakhtiari sheep is one of the most important breeds of sheep in terms of the use of its wool in the textile and carpet industries. In his breed, the dominant coat of the wool is white, although sometimes a percentage of dark brown and pale brown is also observed (Saadat Nouri, M. & Siah Mansour, 1368). However, in this breed, some animals have black spots on their coat, which leads to a decrease in wool quality. Since MC1R, ASIP, KLF4 and MITF genes play an important role in controlling and determining coat color in mammals, the purpose of this study was to investigate the expression of these genes in two phenotypes of white and black spots in the skin tissue of Lori Bakhtiari sheep. Skin samples were obtained from both white and dark parts of 14 white-coated sheep with black spots and total RNA was extracted. The quality and quantity of extracted RNAs were evaluated by agarose gel electrophoresis and spectrophotometer. Extracted RNA samples were exposed to DNase1 enzyme digestion to remove the possible contamination of genomic DNA. Also, the quality of synthesized cDNA was evaluated using 1% agarose gel. In this research, in order to amplify a fragment of the studied genes, using the mRNA sequence of these genes in the GenBank database, appropriate primers were designed by Primer3plus software. To evaluate the relative expression of the target genes, β-actin and GAPDH genes were used as reference genes to normalize the data. Finally, BestKeeper and REST 2009 V2.0.13 software were used for the analysis of gene expression data. Based on the descriptive results of Ct values, MC1R and MITF genes revealed minimum and maximum expression stability among the target genes in skin samples with standard deviations of 1.34 and 3.62, respectively. In addition, the reference genes (β-actin and GAPDH) showed the highest stability among all the studied genes. No significant differences were observed in mRNA levels of MC1R, ASIP, KLF4, and MITF genes in the spotted skin tissue compared to the white part of the skin (p>0.05). However, the expression of the ASIP gene was more than 2 times in the spotted part compared to the white skin, but this difference was not significant (p=0.21). In addition, the MC1R gene showed minimum expression differences in black spots and white parts of the skin tissue. In addition, the MITF and MC1R genes showed the highest and lowest levels of expression in skin samples of Lori Bakhtiari breed sheep with average Ct of 25.86 and 30.42, respectively. However, among all the studied genes, the lowest mRNA level was observed for the GAPDH gene with an average Ct of 35.96. Mammalian coat color results from various factors such as the degree and distribution of melanin pigment and the interaction between genotype and environment (B. Li et al., 2018). In addition, melanogenesis is a complex process that includes melanocyte growth, melanosome formation, melanin synthesis, melanin transport, and melanosome release (Ito & Wakamatsu, 2011). According to the conducted studies, a large number of genes are involved in the mechanism of coat color determination, but two genes, MC1R and ASIP, play an essential role in the regulation and control of coat color (Searle, 1968). In our study, no significant difference was observed in the expression of MC1R, ASIP, KFL4, and MITF genes in the spotted compared to the white part of the skin tissue in Lori Bakhtiari sheep. These results showed that the development of skin spots is not under the control of the studied genes in Lori Bakhtiari sheep, and genes or other factors can play roles in the creation of dark spots in this breed.

Farm animals face different types of abiotic stresses due to many management activities. Stress has adverse consequences on the animal's health and welfare. Laparoscopic artificial insemination (LAI) in sheep is also one of the mini surgery activities that associated with the use of sedatives, catching and fettering the animal, the insertion of instruments into the abdomen, and manipulation of the reproductive tract. All these actions are stressful and may impact stress axis, hematological and biochemical alterations, antioxidant status and fertility outcome. The administration of acepromazine combined with a multivitamin and amino acids complex may be a useful strategy for reducing stress in sheep undergoing laparoscopic AI. In this experiment, 50 non-pregnant Afshar breed ewes, aged 3-4 years, with almost the same body weight and score, were used. The estrous cycle of the ewes was synchronized by flurogestone acetate loaded sponges and eCG hormone. At the time of sponge removal, the ewes were divided into two groups (n = 25). All of the ewes, 54 h after sponge removal, were exposed to stress caused by LAI. In the first group (treatment), with sponge removal, each ewe was received 10 ml of the Multiaminoject intramuscularly. Also, 20 min before LAI, in addition to Multiaminoject, each animal also received 0.0834 mg aspromazine (i.v) per kg of body weight. At the same time, the ewes of the second group were injected with physiological saline and were considered as control. Changes in plasma cortisol concentration and its kinetics were measured from zero (20 min before LAI) to 180 min after through serial blood sampling. Changes of hematological and biochemical parameters of jugular vein blood were evaluated 20 min before and 40 min after LAI. The plasma antioxidant status was measured at the times of sponge removal, LAI and 3 days after LAI. The pregnancy rate was recorded by ultrasound at 45 days. Laparoscopic AI induced stress response in the ewes, by that, 20 minutes after LAI, the concentration of cortisol increased significantly compared to the baseline concentration (P< 0.05). In this research, the injection of the aspromazine along with a multivitamins and amino acids complex could not inhibit the secretion of cortisol during laparoscopic surgery, and it caused the rejection of the hypothesis of this research. The reason for this discrepancy is related to the type of sedative used to reduce or eliminate pain during surgery. It has been reported that plasma cortisol response is reduced by ketoprofen and completely removed by detomidine. As a result, after LAI, the number of white blood cells and the plasma concentration of malodaldehyde increased and the hematocrit, hemoglobin and total antioxidant capacity decreased (P< 0.05), but the concentration of plasma proteins did not change (P> 0.05). Acute stressors cause a transient increase in the number of blood cells in the blood circulation. The cause of this phenomenon is the contraction of the spleen due to the stimulation of catecholamines. In addition, with the increase in cortisol secretion, blood cells are released from the bone marrow into the bloodstream. In a study, the use of supplements containing various vitamins and minerals, one week before and one week after laparoscopic surgery, improved the antioxidant status of patients after surgery and the level of plasma malodaldehyde decreased significantly. The reason for not seeing a significant effect on antioxidant parameters in this research may be the type of supplement containing antioxidants and the time of its use before and after surgery. Injection of aspromazine combined with Multiaminoject increased plasma proteins and decreased the level of aspartate aminotransferase enzyme (P< 0.05). Depending on its intensity and type, stress causes damage and release of tissue and liver enzymes into the blood. In a research, it was shown that the plasma concentration of aspartate aminotransferase and creatine kinase increases due to transportation stress in pigs. The protective effects of vitamin and amino acid supplements in preventing liver damage have been reported in previous studies. The decrease in enzyme concentration after laparoscopy in the treatment receiving aspromazine and multi amino acids may be related to its protective effects. The results of the study showed that there was no significant difference in fertility outcomes between the two groups (P< 0.05). The pregnancy rate for the acepromazine and Multiaminoject-treated group was 50%, while the pregnancy rate for the control group was 45%. This result is probably caused by the lack of interference or partial interference of aspromazine with uterine contractions around ovulation and conception. The authors concluded that acepromazine combined with a multivitamin and amino acids complex, did not have a significant effect on fertility outcome, blood hematological and biochemical alterations and the oxidative stress generated by laparoscopic AI in ewes, but the severity of tissue damage reduce and plasma globulin concentration increase in response to acepromazine combined with a multivitamin and amino acids complex injection. It is important to note that this is just one study and that more research would be needed to confirm these findings.

Sperm cryopreservation is a highly effective technique utilized in reproductive procedures, particularly in artificial insemination, as it enables the transfer of superior genes to the subsequent generation of animals. However, the exposure of sperm cells to low temperatures during the freezing process can have detrimental effects on their integrity, morphology, and viability (Gangwar et al., 2020). To counteract these negative impacts, cryoprotectants are added to the sperm extender. The primary purpose of incorporating cryoprotectants is to fulfill the energy requirements of the sperm, shield them from the adverse consequences of temperature fluctuations, mitigate the physical and chemical stresses associated with cooling, freezing, and thawing, and create an environment conducive to enhancing sperm survival (Barbas and Mascarenhas, 2009). In recent years, plant-based extenders have emerged as a viable alternative to animal-based extenders for semen preservation in various species. Coconut milk, in particular, has garnered attention due to its rich composition of fatty acids (including polyunsaturated fatty acids), amino acids, sugars, antioxidants, vitamins, and minerals. These components not only provide essential nutrients for maintaining cell function but also serve as a suitable medium for sperm extension and culture (dos Reis et al., 2023; Vasconcelos et al., 2009; Yong et al., 2009). Consequently, this research aims to explore the impact of coconut milk on the quality of ram sperm following cryopreservation. Twice a week, semen samples were obtained from five Lori-Bakhtiari rams using an artificial vagina. The collected semen from these five rams was then combined and divided into six different treatments. These treatments consisted of five variations with varying levels of coconut milk (5%, 10%, 15%, 20%, and 25%) and one treatment with egg yolk (15%). Various parameters such as sperm velocity, membrane integrity and activity, morphology, lipid peroxidation rate, and sperm DNA fragmentation were assessed both before and after the process of cryopreservation. The obtained data was subsequently analyzed using the SAS statistical software. Prior to cryopreservation, the results indicated that, with the exception of the BCF parameter, there were no disparities in the various aspects of sperm kinetics, viability, functionality, and morphology. However, a notable decline in sperm motility parameters was observed after thawing across all treatments. This detrimental impact following the freezing process was more pronounced (P<0.05) when using the extender containing coconut milk compared to the extender containing egg yolk. Consequently, in terms of other motility parameters, the egg yolk treatment exhibited significantly higher values (P<0.05) than the other treatments, except for VCL (94.36±10.41%) and BCF (5.83±0.70 Hz). The findings of Wojtusik et al. (2018) were in line with the results obtained in this study. It was observed that when coconut water and coconut milk were used as substitutes for egg yolk in rhinoceros sperm extenders, there was a decrease in both sperm motility and viability. Similarly, the use of powdered coconut water as an extender for cryopreservation of cat sperm resulted in a significant reduction in sperm movement quality compared to a tris extender (de Sousa Barbosa et al. 2020). Furthermore, when comparing different alternatives to egg yolk in the extender of rhinoceros sperm, such as soy lecithin (1 and 2%), coconut water (20%), and coconut milk (20%), it was found that the extenders containing coconut milk or water led to a loss of sperm mobility and viability. However, the extenders containing soy lecithin showed more promise as substitutes for egg yolk, as they maintained sperm viability, morphology, and acrosome integrity better than coconut milk and water (Wojtusik et al., 2018). In contrast to the findings of this investigation, the utilization of extenders comprising powdered coconut water for the cryopreservation of sperm in various animal species, including dogs, boars, horses, goats, and fish, resulted in well-preserved sperm morphological and motility parameters following the freeze-thaw process. Furthermore, studies have demonstrated that freezing goat sperm with coconut milk, bull sperm with coconut oil, and equine and boar sperm with coconut water led to enhanced sperm viability and quality compared to control groups. The ineffectiveness of the extender may be attributed to the presence of salts and ions, such as calcium, potassium, and sodium, in coconut water, which could potentially interact with the channel proteins of the sperm cell plasma membrane. It is important to note that the precise mechanism by which coconut products in the extender maintain sperm characteristics remains unknown. Previous research by Toniolli et al. (1996) suggested that coconut water may serve as a protective agent for sperm due to its content of 3-indoleacetic acid. Furthermore, the replacement of glycerol with dimethylformamide in the boar sperm extender resulted in better preservation of sperm quality after the freezing and thawing process compared to other treatments (Silva et al. 2015). This suggests that each ingredient present in an extender could have a significant impact on enhancing extender efficiency. It is worth noting that coconut milk contains high levels of polyunsaturated fatty acids, which can make sperm more vulnerable to oxidative stress (dos Reis et al., 2023; Vasconcelos et al., 2009). However, no significant differences were observed in the lipid peroxidation indicator and the DNA fragmentation index among the various extenders used in this study. This could indicate that the extenders had similar antioxidant properties. The variations in the reported findings may be attributed to differences in the combinations and quantities of products used in the extenders, the type of base extender employed, variations in semen combinations and the sensitivity of sperm in different animal species, or discrepancies in the coconut combinations utilized (dos Reis et al., 2023). This research suggests that substituting egg yolk with coconut milk entirely in a ram sperm extender does not mitigate the negative consequences of cryopreservation. Additional investigations are necessary to determine the specific composition of the extender incorporating coconut-based ingredients for the cryopreservation of ram sperm.