فصلنامه تحقیقات تولیدات دامی
سال دوازدهم شماره 4 (زمستان 1402)

In recent years, highly pathogenicity avian influenza (HPAI), especially H5N1, has emerged as a major global health concern due to its potential as a zoonotic disease and its devastating impact on poultry populations. Identifying the molecular mechanisms of response to HPAI infection is critical to control, treat, and prevent the risk of a potential pandemic. Microarray technology is becoming a standard technology used in research laboratories all across the world and it is considered as one of the centers of research in cellular processes related to the level and manner of gene expression, including gene function and cell differentiation mechanisms. By using microarray technology, it is possible to obtain a detailed view of the interaction function of genes while simultaneously studying how the genome is expressed. Using microarrays provides the analysis of gene expression in response to viral infections such as influenza, etc., the study of host-pathogen interactions, and also the identification of the effectiveness of drugs and vaccines. This study aimed to analyze the microarray data of H5N1 avian influenza to compare the gene network and analyze the functional pathway in chickens and ducks.

Data mining and searching of microarray data related to Highly Pathogenic Avian Influenza infection was done in the GEO gene expression database (https://www.ncbi.nlm.nih.gov/geo). The microarray data set with accession number GSE33389 based on the GPL3213 platform was selected which contained lung tissue samples challenged with H5N1 virus in chickens and ducks. Normalization of selected microarray data was done using R software, and samples were grouped to compare between infected and control samples. Limma, Biobase, and GEOquery software packages in R software were used to determine the expression level of genes and to investigate the differentially expressed genes (DEGs) between healthy and H5N1 influenza virus-infected lung tissue samples in chickens and ducks. The criterion for selecting significant DEGs was considered as |logFC|>2 and P<0.05. DAVID online tool (https://david.ncifcrf.gov) was used to investigate biological pathways, structural and functional characteristics of genes with different expressions, and functional interpretation of upregulated and downregulated DEGs. It was evaluated and visualized separately based on biological processes (BP), molecular functions (MF), and cellular components (CC). KEGG tool (http://www.genome.jp/kegg) was used to evaluate and study metabolic pathway enrichment. To reveal interactions between proteins and analyze them, STRING database and Cytoscape software were used. While using the Cytohubba plugin to identify and display key genes, the main modules affecting the interaction of genes and proteins were also identified by the MCODE plugin.

Gene expression analysis revealed 2062 and 565 differentially expressed genes between normal and infected tissue in chickens and ducks, respectively (P<0.05 and |logFC|>2). The results of bioinformatics analysis and protein-protein interaction network analysis showed BUB1, NDC80, CDC20, PLK1, PRC1, KIF11, and AURKA genes as hub genes in chicken and also COL6A3, COL3A1, COL4A3, COL18A1, PLOD2, PLOD1, and P4HA2 as highly effective genes in duck (P< 0.05). The results of the ontology comparison of DEGs proved that most of these genes in chickens are involved in the innate immune response and inflammatory resistance of the host, and the most effective genes in ducks play a role in lipid metabolism and energy production to meet the host's resistance to disease. The results of comparative gene network analysis between chickens and ducks are promising to increase our understanding of the host response to H5N1 influenza infection and the factors affecting virus pathogenesis in different avian species. Differentially expressed genes in response to H5N1 infection in chickens and ducks play critical roles in various biological processes, including immune response, inflammation, viral replication, and host-pathogen interactions.In general, gene network analysis showed that chickens and ducks use different genetic strategies to respond to avian influenza virus infection.

The present research was conducted to discover the response to H5N1 HPAI infection in chickens and ducks through comparative gene network analysis. It is important to note that the gene network analysis presented in this research is an initial step towards discovering the response mode of HPAI (H5N1) infection in chickens and ducks, and further functional studies, validation experiments, and integration with other omics data are needed to confirm the role of genes, pathways and hub genes in the host response to H5N1 virus. Therefore, the results of comparative gene network analysis in chickens and ducks obtained from this research can provide valuable insight into the underlying molecular mechanisms of host response to H5N1 influenza infection. Thus, by identifying differentially expressed genes, functional modules, and hub genes in this research, it can be stated that potential targets for future research have been highlighted to some extent. Undoubtedly, further studies in this field will improve our knowledge about the pathogenesis of avian influenza and will help to develop strategies for effective control and prevention of H5N1 influenza outbreaks.

Egg is one of the most valuable food items in the human diet. Several factors, such as diseases, bird age, diet, temperature, humidity, transportation, and storage time can affect the quality of eggs. The longer the storage time, the more CO2 penetrates the eggshell, particularly at room temperature, resulting in decreased internal quality. Eggs are a perishable product and must be stored in the refrigerator from production to consumption. Refrigeration increases production costs and ultimately leads to an increase in the final product price. Currently, there is a growing interest in developing effective methods to preserve the internal quality of eggs. The use of edible coatings after washing the eggs can help preserve the internal quality of the eggs during long-term storage. Edible coatings are an available technology that is currently used to control moisture, gas exchange, and oxidation processes for many products. The use of suitable active and antimicrobial edible films and coatings can potentially reduce the microbial load of egg surfaces, reduce undesirable changes, and increase their shelf life. Proteins are commonly used as film-forming materials. Proteins that have been previously investigated for egg coating development include whey protein isolate or concentrate, zein, and rice protein concentrate. Chicken feathers are a by-product of the poultry industry and are mostly disposed of without any processing, causing severe environmental pollution. To date, hydrolyzed feather protein has not been reported as an egg coating, so this study investigated the effect of hydrolyzed feather protein coating at two concentrations on the shelf life of chicken eggs under room temperature storage conditions.

In this study, a total of 120 fresh white-shelled eggs were obtained from Hy-Line W36 laying hens. The eggshells were washed to remove any surface contamination, then the eggs were numbered and randomly selected for each coating method. The samples were divided into three treatments of 40 eggs each treatment in a completely randomized design. The treatments included control (0%, without coating), a coating containing 10% (w/v) hydrolyzed feather, and a coating containing 20% hydrolyzed feather. Pure glycerol (Serva, Germany) was added to hydrolyzed feathers (2% w/v). The feather hydrolyzed protein was prepared at Tarbiat Modares University using alkaline hydrolyzed raw chicken feathers obtained from a commercial broiler slaughterhouse (Iran Borchin, Tehran). The chemical analysis of hydrolyzed feather protein was performed based on standard methods (dry matter, protein, ash, and fat), and the amino acid profile of the protein was also tested. Some nutritional or toxic important minerals were also measured. Egg weight, Haugh units, albumen weight, yolk weight, yolk color, albumen pH, yolk pH, and shell strength were measured during storage weeks 0, 1, 2, 3, and 4.

According to the data of this study, the height of the egg white and Haugh units in the stored eggs were significantly affected by the experimental coatings (P<0.05). The height of the egg white and Haugh units were significantly higher in the treatment with 20% hydrolyzed protein coating compared to other treatments (P<0.05). The yellowness of the egg yolk was significantly affected by the experimental coatings in the third week (P<0.05). The highest egg quality score was observed in the first and third weeks in eggs with 20% hydrolyzed feather protein coating (P<0.05). The eggshell characteristics were not affected by the experimental coatings. The egg white and yolk percentages were not affected by the experimental coatings. The pH of the egg white in the first and third weeks decreased significantly in eggs with 20% hydrolyzed feather protein coating compared to other treatments (P<0.05). The pH of the yolk was not affected by the experimental treatments. The pH in both the yolk and egg white increased over time. Eggs with coating had significantly less weight loss compared to uncoated eggs (P<0.05). The lowest weight loss was related to eggs with 10% and 20% hydrolyzed feather protein coating, which had a significant difference with uncoated eggs (P<0.05). The weight loss after seven days of storage in uncoated eggs (2.12%) was almost double the weight loss in coated eggs (1.16%). The weight loss after 14 days of storage at room temperature in uncoated eggs (4.21%) was almost double the weight loss in coated eggs (2.35%). The weight loss after 21 days of storage at room temperature in uncoated eggs (6.8%) was almost double the weight loss in coated eggs (3.35%). The weight loss after 28 days of storage at room temperature in uncoated eggs (8.27%) was almost double the weight loss in coated eggs (4.80%). Based on the results of this study, it was found that the use of hydrolyzed feather protein coating can improve the internal quality of eggs during the storage period. For future research, it is recommended to conduct further studies on the effect of hydrolyzed feather protein coating on the quality characteristics of eggs during storage. Previous studies have been conducted on improving the internal quality of eggs during storage using protein coatings with different hydrolysis. Some of these studies have shown that protein coating with hydrolysis can significantly improve the quality characteristics of eggs. Additionally, some studies have been conducted on the effect of protein coating using different proteins such as rice protein, meat protein, whey protein isolated, or whey protein concentrate and zein.

There was no significant difference in weight loss between 10% and 20% hydrolyzed feather protein coverage in different weeks, but the 10% hydrolyzed feather protein coverage provided better protection against weight loss. Since weight loss is mainly due to eggshell water evaporation, it was observed that despite being thinner, the 10% hydrolyzed feather protein coverage provided better protection compared to the 20% hydrolyzed feather protein coverage. Therefore, the use of coverages during egg storage can increase shelf life while maintaining egg quality.

Probiotic bacteria are Gram-positive and negative-catalase with various characteristics including resistance to acid and bile salt conditions, antimicrobial characteristics, bacitracin production, and lack of capability for transferring genes resistant to antibiotics. These bacteria are a part of selected and useful bacteria in the digestive system, which leads to reinforcement of the body's immune system if they are adequately consumed (106-107 CFU/g). This research aimed to identify molecular identification and assessment of probiotic characteristics of lactic acid bacteria (LAB) isolates separated from the digestive and reproduction system of  Ross 308 broiler breeders.
Twenty Ross 308 broiler breeders were selected and samples of the vagina and ileum of them, and the cecum and feces of roosters were taken to separate LAB. The Gram and catalase test was used to approve the biochemical characteristics of LAB. The suspension containing each LAB isolate was harvested at 4  for five min (10,000×g) to assess the survival of the selected LAB isolated under conditions simulating the GI tract. Then, remained sediment in the buffer solution containing HCL (1N) reached to pH equal to two by eliminating the supernatant. After adding 0.1 % (w/v) pepsin to bacterial suspension, it was stored at 37  for three hours. After incubation, the pH of suspension with NaCl (1N) was reached to six. Then, tolerance to small intestine condition was evaluated in PBS solution (pH=7), containing Oxgall (0.3% w/v) and pancreatin (0.1% w/v) of washed suspensions of the LAB was mixed and incubated at 37 . Finally, the LAB isolate population was determined by consecutive dilution in sterile PBS and plate media on MRS agar compared to a blank sample (untreated). For molecular identification of dominant lactic acid bacteria, the DNA of the dominant lactic acid isolate was extracted by polymerase chain reaction (PCR) kit. Then, it was amplified using primers (44F; 1542 R) in temperature conditions. Afterward, for initial approval, PCR products were transferred to 1.5 % agarose gel and electrophoresis was performed in TBE buffer in the presence of positive control and negative control samples. The good diffusion method was used to determine the anti-bacterial effect of the selected LAB isolated against pathogenic factors. To assess auto-aggregation of the selected isolate, the cells obtained from its 24-hour culture were separated by refrigerated centrifugation (10 min, 4 , 6000×g) and was dissolved in phosphate buffer during two phases (pH=7.2) so that the obtained suspension had absorption equal to . Then, the suspension was put at the temperature of 37  for four hours. Then, the absorption of LAB isolate suspension in 600 nm was read. A combination of an equal volume of LAB isolates suspension and pathogenic bacteria were vortexed and incubated at 37℃ for four hours to assess auto-aggregation of isolate. The surface part of the suspensions was read at 600 nm and calculated. About 200 µl of 24 h culture of selected LAB was added to four mL of 1 % MRS agar medium to evaluate the selected LAB isolated antibiotic susceptibility. This combination was overlaid on plates containing 1.5 mL of 1.5% MRS agar, and then, discs of antibiotics including Ampicillin, Gentamicin, Streptomycin, Cefazolin, Ciprofloxacin, Penicillin, Cephalothin, Imipenem, Novobiocin, Clindamycin, Vancomycin, Ceftriaxone, and Nalidixic acid was placed on each plate. After 24 h of incubation at 37℃, the diameter of the inhibition zone (mm) was measured and reported as resistant, relatively sensitive, and sensitive. To consider the capability of hemolysis of blood, LAB isolate was streaked on the surface of a blood agar plate supplemented with 5% sheep blood. After 48 h of incubation at 37℃, the plates were considered in terms of diameter inhibition zone creation and color change in the medium.
The results of the sequencing test led to the identification of Levilactobacillus brevis. The predominant LAB isolated from the gastrointestinal tract (GIT) and reproductive tracts of Ross 308 broiler breeders and L. brevis isolated from the ileum (82.00%) (P<0.05) in the simulated conditions of the GIT compared to the L. brevis isolated from other parts GIT and the reproductive system had more survival than acid and bile. Examining the anti-bacterial effect of the aforementioned isolate showed that the L. brevis bacterium had an inhibitor influence on Shigella dysentery bacteria (87.00%) and Staphylococcus aureus (81.25%). In addition, the highest and the lowest characteristics of co-aggregation of this isolate were observed against Listeria monocytogenes (46.00%) and Salmonella typhimurium (38.00%). This isolate had 43% auto-aggregation characteristics and no hemolytic activity. Assessing antibiotic resistance of LAB isolate showed that the biggest diameter of inhibition zone of bacterium (23.5 mm) was related to Imipenem that had no significant difference with Ampicillin (22.5 mm) and Vancomycin (22.5 mm) and its lowest value (12 mm) was related to Ceftriaxone that had no significant difference with Gentamicin and Cephalotin. The results of this study showed that L. brevis bacterium can be used in the nutrition of broiler breeders as a probiotic bacterium.
The use of probiotics in the feeding of Ross 308 broiler breeders may eliminate the public health concerns of antimicrobial resistance development to some extent, as this could replace the use of antibiotics. According to antimicrobial characteristics, antibiotic resistance, hemolytic activity, auto-aggregation, and co-aggregation of predominant LAB isolate, it can be concluded that L. brevis can be useful and applicable as a probiotic supplement in producing food and pharmaceutical products for broiler breeders.

Based on current knowledge, it has been proven that many edible ingredients in addition to meeting the needs of creatures, in terms of energy and protein, contain compounds that affect cellular actions and intracellular signaling pathways and can temporarily or permanently change the function of the cell. The use of molecular biology tools and genetic research defines the mechanisms through which gene expression is affected by food, and conversely, these genes also affect the absorption of food, metabolism, and excretion. From the point of view of nutrigenomics, dietary nutrients are signals that are received by sensitive cell systems and can affect the expression of genes and proteins and the production of metabolites. Nutritional manipulations and strategies are key tools to influence ruminant production. Fatty acids act on the nucleus by binding to and regulating the activity of specific nuclear receptors or transcription factors, thus playing a central role in regulating the expression of genes involved in fatty acid uptake by muscle cells. Interactions between diet nutrients and the expression of genes involved in lipid metabolism have many possibilities regarding the deposition of fatty acids in the tissue. The study of gene expression has enabled clarification of the mode of fatty acid metabolism in muscle and the accumulation of intramuscular fat or marbling and the role of the genes that promote fatty acid oxidation and mitochondrial respiration in the liver muscle and adipose tissue. Polyunsaturated fatty acids with multiple double bonds (PUFA) and monounsaturated fatty acids with one double bond (MUFA) have received much attention in the last decade, and their health benefits are increasingly evident. Therefore, this study investigated the effect of using calcium salts of fish oil, olive oil, and saturated fat on the expression of some genes related to fat metabolism in Lori Bakhtiari×Romanov fattening lambs.
This study was carried out at the educational research station of the Department of Animal Science of the College of Agriculture and Natural Resources, University of Tehran. 49 male lambs aged four to five months with an average initial weight of 29.97 ± 0.88 kg were divided into seven groups of seven lambs in a completely randomized design. The experiment consisted of seven treatments with a basic diet as follows: 1) Basic diet without fat powder (control), 2 and 3) Basic diet with calcium salt of fish oil (rich in omega-3 fatty acids) in the amount of 2% dry matter of the diet for 90 and 45 days, respectively, 4 and 5) Basic diet with calcium salt of olive oil (rich in omega-9 fatty acids) at the rate of 2% dry matter of the diet for 90 and 45 days, respectively, 6 and 7) Basic diet with saturated fat powder in the amount of 2% dry matter of the diet for 90 and 45 days, respectively. Rations were adjusted based on the NRC Sheep and Goat and using the fifth version of CNCPS (The Cornell Net Carbohydrate and Protein System) software so that they are the same in terms of energy and protein. Twenty-eight lambs were slaughtered and a liver sample was taken for nutrigenomics studies. Total RNA was extracted from liver tissue samples using the RNA extraction kit produced by Dena Bio Asia Company (Mashhad) and according to the protocol provided with the kit. The expression of four main genes involved in lipid metabolism in liver tissue was investigated to provide comprehensive information on the effects of omega-3, omega-9 fatty acids, and saturated fat at the molecular level.
The results showed that feeding with calcium salts of fish oil, olive oil, and saturated fat, both in the whole 90 days or the last 45 days of the fattening period, had no significant effect on the expression of FADS1 and FADS2 genes in liver tissue compared to the control group. However, the use of calcium salts of fish and olive oils in periods of 90 and 45 days significantly increased the hepatic mRNA expression of the CPT1 gene (P=0.001) and ACOX1 gene mRNA expression in liver tissue compared to control and saturated fat treatments (P=0.002). The CPT1 enzyme is responsible for a mitochondrial transport system and plays a key role in controlling the oxidation of long-chain fatty acids and stimulating mitochondrial β-oxidation.
The results of this study showed that diet supplementation with calcium salts of fish and olive unsaturated fatty acids, regardless of the period of consumption, increased the expression of genes involved in the lipolysis of liver tissue fats of lambs.

The lack of animal feed resources and the increase in animal feed costs relative to the total production costs have created challenges in supplying society with animal protein. Therefore, correctly estimating the nutritional value of feed, especially protein sources, can be an important step in meeting the needs of livestock and reducing production costs. Protein is the most important and expensive nutrient used in ruminant diets. Using non-protein nitrogen (NPN) sources such as urea or its products can be considered a cheap alternative source in animal feeding. Iran is one of the most important urea-producing countries in the world; therefore, using urea or its slow-release products as a feed additive in animal nutrition can be beneficial. In the past years, various sources of slow-release urea have been produced and used in animal feeding. Biuret (heated urea) is one of the slow-release urea compounds, and its production cost is lower than other slow-release urea sources. Compared to urea, it has less solubility in water and is converted to ammonia in the rumen at a much slower rate. It is also less toxic than urea and has a lower negative effect on feed palatability, so it can be used to a greater extent than urea in the diet of ruminants. Biuret is very safe and it can be included in the diet of animals in an amount of 20 times higher than the toxic dose of urea. Biuret usually contains 248.5% crude protein (39.8% nitrogen content), which is a little less than urea. On the other hand, increasing the daily feeding frequency is one of the management methods that may improve the productive performance of livestock, as well as the quantity and quality of the carcass. Increasing the feed frequency may improve nitrogen retention and reduce body fat. It is believed that increasing the frequency of daily feeding will keep the feed fresh, reduce the amount of feed waste, and improve livestock performance. Therefore, this study aimed to investigate the effect of NPN source, in high-protein diets, and feeding frequency on growth performance, rumen parameters, and the activity of rumen microbial enzymes of fattening lambs.
Twenty-eight male Lori-Bakhtiari fattening lambs (age: four to five months; live weight: 36.1±3 kg) were assessed for 60 days using a 2×2 factorial experiment in a completely randomized design with four treatments and seven replications. The experimental high-protein diets (16% of DM) contained urea or biuret, which were given to the lambs in the form of a total mixed ration two or three times a day. The lambs were kept in 1×1.5 m2 individual stalls. Nutrient intake, growth performance, and ruminal parameters [on day 45 of the experiment at fasting (before morning feeding), three, and six h after feeding] including pH, ammonia and volatile fatty acid concentrations, and microbial enzyme activity were measured.
Results showed that intakes of dry matter, organic matter, crude protein, neutral detergent fiber and ether extract, final weight, and total weight gain were not affected by NPN type or feeding frequency (P<0.05). Compared to urea, lambs consuming biuret had higher average daily gain and better feed conversion ratio (P<0.05). The ruminal pH was not affected by the NPN source, feeding frequency, and their interaction (P<0.05). Rumen ammonia nitrogen concentration at three and six hours after morning feeding was significantly higher in lambs fed the diet containing urea than in biuret (P<0.05). The effect of NPN source, feeding frequency, and their interaction was not significant at 0 and six h after feeding on the ruminal concentration of total volatile fatty acids (VFA), acetate, propionate, butyrate, valerate, iso-valerate, and acetate to propionate ratio (P<0.05). However, feeding a diet containing biuret and given three times a day increased total VFA and acetate concentration at three hours post-feeding (P<0.05). The ruminal activity of carboxymethyl cellulase at 0, three, and six hours after feeding increased in animals fed biuret-containing diet compared to urea (P<0.05). However, rumen protease activity significantly increased in lambs fed a urea-containing diet than biuret-containing diet (P<0.05). Feeding three times a day increased the activity of carboxymethyl cellulase at three hours post-feeding (P<0.05). Filter paper degrading activity at three and six hours after feeding in lambs fed biuret was significantly higher than those fed urea (P<0.05). The activity of microcrystalline cellulase and α-amylase was not affected by the NPN source, feeding frequency, and their interaction (P<0.05).
Using biuret compared to the urea, in high-protein diets, and feeding diets three times a day, compared to two times a day, improved rumen fermentation parameters and performance of fattening lambs.

The management of suckling calves guarantees health and performance in their productive life. The sensitivity and low immune system in newborn calves increase the odds ratio of some disorder incidence and sometimes it is associated with the death. Therefore, the use of antibiotics in suckling calves became popular. Laws prohibiting the use of antibiotics in raising domestic animals led to the use of natural alternatives with similar properties. These compounds cause positive effects on the reduction of intestinal infections, and disorders and increase the absorption of nutrients by creating intestinal microbial balance. Prebiotics have the same feature that changes the microflora population in the digestive system. It has been proven honey has prebiotic characteristics that improve the immune system in mammals. This product and its by-products are used for different goals in human feed. This prevents intestinal infection, modifies intestine microflora population, and improves health situation and lipid metabolism. Honey is a natural product that is thickened from saturated or supersaturated sugar solutions. It usually consists of 17% water, 38% fructose, 31% glucose, 10% other sugars, and a wide range of micronutrients, vitamins, minerals, and amino acids, with a pH below 4. This study was conducted to evaluate the effect of milk enrichment with natural honey on the performance, digestibility, blood parameters, and skeletal growth indices of suckling Holstein calves.
Eighteen Holstein suckling male calves with an average weight of 58±4.2 kg were used. Treatments were divided into three groups which contained zero, 2.5, and five g per day of natural honey to the milk consumed. The duration of the experiment was 30 days evaluated in a completely randomized design. Calves were kept in individual pens and fed based on NRC requirements. Starter provided to total mixed ration (TMR) form which is given at 8:00 am and 4:00 pm daily. Calves fed milk based on 10% of body weight in the morning and evening. The amount of milk consumed by calves is measured, and to calculate the milk solids, it is multiplied by a coefficient of 12.5%. Dry matter (DM) digestibility, blood samples, average daily gain, dry matter intake, stool score, and skeleton growth were evaluated during 30 d and compared treatment by the general mixed model in SAS software.
The results of this study showed that adding natural honey to milk significantly improved the feed conversion ratio and increased the final weight, average daily gain, and dry matter digestibility of the calves (P<0.05). The group of calves that consumed five g of natural honey per day had the highest final weight and average daily gain, as well as the lowest feed conversion ratio. However, the milk and starter intakes were not affected by the experimental treatments, and there was no significant difference observed between the control group and the groups that received natural honey (P<0.05). Supplementing milk with honey improved the growth rate and feed conversion ratio of the calves, likely due to the presence of enzymes and other substances in honey that aid in breaking down polysaccharides into usable energy for the animals. Blood glucose concentration decreased in the calves that received natural honey (P<0.05), but the concentrations of cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein were not significantly different among the treatments (P>0.05). The reduction in blood glucose can be attributed to the antioxidant compounds present in honey, which can reduce intestinal glucose absorption by inhibiting the digestive enzymes of alpha-amylase or delaying the emptying of stomach contents into the small intestine. The stool consistency score increased with increasing levels of natural honey in the milk, and the group that received five g of natural honey per day had the best stool score, while the control group had the lowest stool consistency score (P<0.05). However, there was no effect of treatment on the number of animals with diarrhea or the number of days involved with diarrhea (P>0.05). Natural honey is rich in antioxidants and has prebiotic properties, which promote the growth of beneficial microorganisms and decrease harmful microflora in the gut. There was no significant difference observed in skeletal growth indices between the different experimental treatments (P>0.05).
The experiment's findings indicated that incorporating natural honey (up to five g/day) into the milk given to Holstein calves can improve their performance and health, making it a recommended practice.

The assessment of commercial silkworm hybrids based on cocoon characteristics will provide only profit to the sericulturists. However, the profits of sericulturists and silk spinners can be guaranteed by paying more attention to quality and quantity characteristics in both cocoons and fibers. In silkworms, the paternal and maternal lines of commercial hybrids are crossed reciprocally. That is the reason that hybrids of silkworms raised by sericulturists are not so identical. This genetic difference causes a difference in the final quantity and quality of the productive cocoon and silk thread. This study aimed to compare commercial silkworm hybrids based on cocoons and silk thread performance of Guilan sericulturists. In this study, the silk filament characteristics of 12 imported silkworm hybrids and two domestic hybrids were compared in two regions of Guilan province. The weight, size, length, and strength (Tensile strengths and Elongation percentage) of the silk filament in addition to the raw silk percentage were investigated. The cocoon characters were the performance of each box, cocoon weight, cocoon shell weight, cocoon shell ratio, the percentage of good cocoons, the number of cocoons per liter, pupal mortality, cocoon length, and cocoon width. Statistical analysis was done using the GLM procedure of SAS software. The hybrids from reciprocal mating were compared by t-test. To clustering, the WARD method was applied based on the deviation from the standardized number (Z-score) using the SPSS software. Variance analysis of traits showed that geographical region has a significant effect on thread diameter (P<0.05). The highest silk filament weight was related to hybrid 871×872, and two Iranian hybrids (154×153 and 104×103) with 5.01, 4.89, and 4.88 grams, respectively, and the lowest for BxQ hybrid with 3.57 grams. For filament diameter, the Iranian hybrids (104×103 and 154×153) had the highest filament size with 69.63 and 61.05 deniers, respectively. Higher diameter in Iranian silk filament fits the silk thread consumption type in Iran. The silk thread in Iran is used to produce silk carpets and rugs, therefore, it needs a larger thread diameter than when the goal is to produce high-quality silk fabrics that require fine thread. The Iranian hybrid 154×153 was excellent in terms of all the important features of the silk filaments, including strength, weight, diameter, and length. M×S, HB×JA, and B×Q hybrids had the smallest thread diameter with 53.72, 54.60, and 54.76 deniers, respectively. Hybrid Q×B was superior to some hybrids but for reciprocal hybrid (B×Q), the overall performance was not favorable. This hybrid had the lowest yield of silk filament weight, diameter, and length. A comparison of hybrids resulting from reciprocal crossing showed that the famous hybrids Q×B and B×Q had the highest differences among six-pair imported hybrids. The difference was observed between the two hybrids for six characteristics including cocoon shell weight, cocoon weight, the number of cocoons per liter, the percentage of good cocoons, the weight and size of the silk filament, and the raw silk percentage. It certainly affects the profits of both sericulturists and silk spinners. The classification of hybrids based on the WARD method led to three groups and the Iranian hybrids were in one group. Q×B and B×Q hybrids and their rejuvenated hybrids (BB×QA and QA×BB) were in the third group. M×S and S×M along with 871×872 and 872×871 hybrids were in the same group. Therefore, if necessary replacing hybrids within the group will be possible. The cocoon production of imported hybrids showed that there is a big difference between the performance mentioned in the catalog and what was produced under the conditions of rural silkworm rearing in Guilan province. Elongation percentage,  raw silk percentage, filament length, cocoon width, pupal mortality, cocoon weight based on total good cocoons, and number of cocoons per liter had no significant difference between domestic and imported hybrids. Due to the better performance of BB×QA and QA×BB, especially the similarity of reciprocal hybrids, these rejuvenated hybrids can replace the old ones (Q×B and B×Q). M×S and S×M hybrids, which had the advantage of uniformity between direct and reverse hybrids, can be considered more due to more silk filament weight and raw silk percentage. The results of this study showed that the most important factors in silkworm rearing are the performance of the silkworm box and the raw silk percentage to be improved for Iranian hybrids.