فصلنامه تحقیقات تولیدات دامی
سال یازدهم شماره 4 (زمستان 1401)

To be successful in rearing newborn calves, it is necessary to maximize feed efficiency and health status. One way is to use feed additives such as probiotics. Probiotics reduce the harmful microflora of the digestive tract (such as coliforms) and metabolic and infectious diseases and improve beneficial microbes, the defense system, nutrient absorption, feed consumption, and animal growth. However, the animal response to probiotics is not uniform. Therefore, if the conditions for the activity of probiotics in the digestive system are improved, it may be possible to increase the effectiveness of these microbial additives and achieve a more uniform and reproducible response. For this purpose, it may be possible to use biochar in diets containing probiotics. Biochar can absorb organic substances and gases, bind toxins, and provide a favorable environment for useful microorganisms, increasing fermentation efficiency and livestock performance. However, few findings are available on the effect of biochar, Saccharomyces boulardii (yeast), and different commercial lactobacilli mixtures in young calves. We hypothesized if biochar is included in diets containing probiotics, it could probably provide better conditions for the probiotic activity in the digestive system and thus improve the response of the calves to these additives. Therefore, this research investigated the effect of adding biochar (pomegranate and plum woods) in diets containing lactobacilli (L. plantarum, L. rhamnosus, and Enterococcus faecium mixture) and yeast (S. boulardii) on in vitro fermentation, microbial population, methane release, antioxidant capacity, as well as health indicators, rectum bacteria and blood enzymes of the pre- and post-weaning calves.
The experimental treatments were: 1. Basal diet without probiotics and biochar (control), 2. Diet + lactobacilli mixture, 3. Diet + S. boulardii, 4. Diet + biochar, 5. Diet +lactobacilli-biochar, and 6. Diet + S. Boulardii-biochar. The in vitro experiment was conducted with three replicates and two different runs. Digestibility coefficients were determined using Tilley and Terry method. A gas production technique was used to assess truly digestible substrate (TDS), partitioning factor (PF), microbial biomass production (MBP), cellulolytic bacteria, total protozoa, methane release, and antioxidant capacity. The in vivo experiment was done using 60 newborn Holstein calves (six treatments and 10 replicates) in a randomized complete block design. The start of the experiment was at the age of 10 d, weaning at 75 d, and the end of the experiment at 100 d. The daily intake and growth of the animals were recorded. In pre- and post-weaning calves, the rectum bacteria (coliforms, lactobacillus, and total aerobics), urinary and fecal pH, health score criteria (including scores of feces, nose, eye, ear, cough, temperature, navel, and joint), and total average health score were determined using standard methods. Moreover, the blood serum enzymes (alkaline phosphatase, Gamma-glutamyl transferase, aspartate aminotransferase, alanine transaminase, and lactate dehydrogenase) were assessed. Data were analyzed using the MIXED procedure of SAS.
Separate inclusion of the lactobacilli mixture, S. boulardii, and biochar in the diet improved in vitro digestibility, TDS, cellulolytic bacteria population, and antioxidant capacity compared to the control group (P<0.05) with maximum improvements when probiotic-biochar mixtures were used. The MBP and PF were improved by including the additives in the diet. Probiotics and probiotic-biochar mixtures decreased protozoa (P<0.05). Different additives decreased methane production, and the least methane was observed in diets containing lactobacilli-biochar and yeast-biochar (P<0.05). Probiotics provide better conditions for the growth and activity of appropriate microorganisms. In addition, the highly porous structure and high surface area of biochar increased the establishment, attachment, and growth of useful microbes. These properties improved cellulolytic bacteria, organic matter degradation, and MBP. The improved antioxidant status could be due to the effect of probiotics in eliminating oxidant compounds in the digestive system, and the activity of biochar in absorbing toxins and unfavorable factors in the fermentation environment. As a result, the simultaneous use of probiotics and biochar improved in vitro fermentation variables more effectively. The reduction of methane can be due to the decrease of protozoa and methanogens, and the increase of methanotrophs. The results of the second experiment (in vivo) showed the probiotics and biochar reduced the rectum coliforms and improved (reduced) the fecal score and average health score of pre- and post-weaning calves so that the greatest improvement was seen in probiotic-biochar treatments (P<0.05). Cough and body temperature scores also tended to improve by feeding additives. Alanine phosphatase, gamma-glutamyl transferase, aspartate aminotransferase, and alanine transferase were not affected by the treatments. However, lactate dehydrogenase in the pre-weaning period was lower in the additive treatments compared to the control group (P<0.05), and the minimum value was observed in probiotic-biochar groups. The use of probiotics can improve the rumen development, ruminal and intestinal population of useful microbes, and prevent diarrhea, which results in enhancing digestion, passage rate, feed consumption, and animal performance. Moreover, adding biochar enhances digestibility and microbial growth, binds toxins, reduces energy loss, and induces a suitable environment for beneficial microbes, resulting in better animal performance. The reduction of coliforms may be due to the binding of probiotics to the digestive system wall, nutrient competition, preventing harmful microbes, improving digesta flow, and changing the pH of the digestive tract. Moreover, biochar provides a suitable growth environment for beneficial microbes, removes toxins and unwanted substances from the digestion environment, reduces viscosity, and probably makes the digestive system unsuitable for harmful species including coliforms.
Separate inclusion of the lactobacilli mixture, S. boulardii, and biochar in the diet improved in vitro fermentation variables and the health status of pre and post-weaning Holstein calves, without negative effects on blood enzymes. The best in vitro and in vivo responses were observed when probiotics and biochar were used simultaneously. Therefore, the addition of biochar (1% of DM) to diets containing probiotics (lactobacilli and yeast; 2 g/d) can be recommended as a strategy to enhance the effectiveness of the probiotics in calves and to reduce environmental pollution due to the decreased methane emissions.

The use of highly concentrated rations in the fattening of ruminant animals is done to improve the performance per unit of feed consumption. Increasing the amount of concentrate in the ration improves the food conversion ratio and the weight gain of fattening lambs, and the production of volatile fatty acids. However, some in vivo experiments have reported that long-term use of highly concentrated diets is not always associated with increased performance. Therefore, feeding diets containing high amounts of concentrate (with a limited amount of effective fiber) often leads to metabolic disorders, the most important of which is subacute ruminal acidosis which causes a decrease in dry matter consumption and reduces the fiber digestibility, milk fat, lameness, and liver abscess, and even leads to the death of the animal. Moreover, sub-acute acidosis leads to significant economic losses. Therefore, preventive measures to prevent acidosis and improve starch digestion, such as the use of antibiotics, probiotics, and buffers, have been taken into consideration. Lactate-consuming bacteria metabolize lactic acid and control its accumulation in the rumen. This process is important when the animal diet contains a large amount of grain. Megasphaera elsdenii and Selenomonas ruminantium are the dominant strains that consume lactic acid in the rumen, and among these two strains, Megasphaer alsdeni consumes 65 to 95% of the lactate in the rumen. The use of biological methods in controlling pH changes can also reduce the dependence of livestock on chemical additives to some extent. Therefore, the present experiment was conducted to compare the effect of different chemical and biological buffers (acid-consuming bacteria) on the digestion and fermentation of highly concentrated diets.
The experimental treatments included: 1. The control diet (without additives and containing 70% concentrate + 30% fodder or basal diet), 2. Basal diet + 3 mL of bacteria Megasphaera elsdenii (bacteria- 4.5 × 108 cfu/3mL), 3. Basal diet + 1% sodium bentonite, 4. Basal diet + 1% sodium bentonite + 3 mL of bacteria, 5. Basal diet + 1% sodium bicarbonate, 6. Basal diet + 1% sodium bicarbonate + 3 mL bacteria, 7. Basal diet + 1% magnesium oxide, 8. Basal diet + 1% magnesium oxide + 3 mL bacteria, 9. Basal diet + 1% zeolite, 10. Basal diet + 1% zeolite + 3 mL bacteria, 11. Basal diet + 1% sodium sesquicarbonate, and 12. Basal diet + 1% sodium-sesquicarbonate + 3 mL bacteria. Megasphaera elsdenii bacteria were isolated and prepared from Najdi goats at the Agricultural Sciences and Natural Resources University of Khuzestan (Ahvaz, Iran) and their activity was investigated in experiments. In gas production experiments, the experimental samples were ground with a mill containing a 1 mL sieve. About 200 mg of the dry matter of the desired sample was weighed and poured into 100 mL vials, and eight replicates were considered for each treatment. The produced gas of the samples was recorded at 0, 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours of incubation using a digital barometer.
The effect of experimental treatments on potential and rate of gas production, partitioning factor, microbial biomass production, microbial biomass production efficiency, pH, ammonia nitrogen concentration, apparent dry matter digestibility, and protozoa population was significant (P<0.05), and except for ammonia nitrogen, all parameters in buffer treatments were higher than the control. The highest gas production potential (68.26 mg), microbial biomass production (1212.31 mg), and microbial biomass production efficiency (79%) were observed in the treatment containing sodium bicarbonate + bacteria (P<0.05). The highest pH and ammonia nitrogen were for the treatment containing bacteria (6.60) and control (27.30 mg/100 mL). The total protozoa population was the highest in the treatment of sodium bentonite + bacteria.
In general, the results of the present experiment showed that the use of buffers improved digestion and fermentation conditions, and each of the buffers had a greater effect on one or more parameters than the others. In addition, acid-consuming bacteria as a pH regulator had competitive effects with chemical buffers, especially bicarbonate buffer, and even better in some cases. The effect of some chemical buffers used in the present experiment, such as bicarbonate, has been further investigated, but the effect of other chemical and biological buffers mentioned in the present experiment on the digestive and fermentation properties of ruminant animals has been investigated less. Therefore, it is recommended to investigate the effect of chemical buffers used in the present experiment alone or together with acid-consuming bacteria in feeding ruminant animals.

The profitability of dairy cattle herds mainly depends on the reproductive performance of cows. Because reproductive performance is effective on the amount of daily milk and calf production per cow, increasing the number of inseminations per conception, lengthening the calving interval in the herd, and causing the voluntary (due to the age of the animal) and involuntary (due to illness or fertility disorders) elimination. For evaluating fertility traits, we sometimes come across domains whose records were incomplete at the time, out of range, or delayed. These records are called censored records. The typical way to deal with such records is to delete them from the data set. However, this reduces the information available for genetic evaluation and may affect trait variance by masking actual genetic differences between animals. Another way is to use appropriate statistical methods to place censored records in genetic evaluations. The use of censored records in genetic evaluations yields more reliable genetic parameters, and it can increase the estimation accuracy of breeding values giving the support that the breeding value estimation is close to the true breeding value. The present study aimed to investigate the role of censored records in the estimation of genetic parameters of the number of inseminations per conception in Iranian dairy cows.
The dataset included 29621 records collected from 2005 to 2019 by the Vahdat Cooperative of Isfahan in Iran. FoxPro software (version 9.0) was used to edit and prepare data, remove incorrect records and create contemporary groups. Five animal models, including Threshold Model (TM), Penalty Model (PM), Modified Penalty Model (MPM), Threshold-Threshold Model (TTM), and Modified Threshold-Threshold Model (MTTM), were used for the genetic evaluation of this trait. The predictive ability of the models was assessed using cross-validation. Also, a simulation study was performed to determine the appropriate model.
The estimated heritability using different models was 0.3 (0.013), 0.07 (0.013), 0.07 (0.013), 0.3 (0.13), and 0.4 (0.012) for TM, PM, TTM, MPM, and MTTM, respectively. The highest correlation of breeding values for all animals was between MPM and TM (0.98). The same result was obtained for the best sire (sire with at least 20 daughters). The high correlation shows that these models work similarly in selecting the best sires. The low correlation between models indicates that the models rank sires differently. Therefore, the choice of model can affect the ranking of males. The top 10 sires showed less correlation between models than the active sires. When the number of males decreases, the selection becomes more strict. The prediction accuracy in models with censored records is higher than in models without censored records because the use of models that include censored records is due to the increased information about the trait, and the absence of bias due to deleting records improves the accuracy of estimating breeding values. Consequently, if the censoring status is not taken into account, animals with superior breeding values may be biased. The low ratio of common superior animals in different percentages between models showed that these two models rank the animals differently. Still, the high proportion of common superior animals indicates the similarity of the two models in choosing the best animals. So, model selection can be effective in ranking superior animals because when the censored record is used, the information is increased and the situation would be closer to reality. The cross-validation showed that the accuracy of the breeding value prediction by different models was higher in models with censored records than in models without censored records. The results of the simulation study showed that the models with censored records provided better genetic parameter estimates. TTM has been able to estimate expected heritability better than other models. The percentage of top animals in common between the top 10 and the top one percent of simulated animals with different models showed that the ability of the TTM model to select real top animals is more than other models, which shows that this model has identified top animals better.
The results of the present study showed that the censored records are important for estimating the genetic parameters of the number of inseminations per conception in Iranian dairy cows and ignoring those led to a biased estimation of the parameters. Data simulations also showed that using censored records provided more realistic genetic parameter estimates.

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. Selection for higher litter size in sheep has led to a variation of this trait within and across different breeds. Natural and artificial selection related to adaptation and economic traits, such as litter size, results in changes at the genomic level which leads to the appearance of selection signatures. Detection of these regions provides an opportunity for a better understanding of genetic mechanisms underlying the phenotypic variation of litter size in sheep. Several tests including the linkage disequilibrium-based approach, site frequency spectrum, and population differentiation-based approach have been developed to explore the footprints of selection in the genome. This study aimed to identify selection signatures in Baluchi sheep to investigate the genes annotated in these regions as well as the biological pathways involved. For this purpose, XP-EHH and FST analyses were conducted using the genome-wide single nucleotide polymorphisms (SNPs).

In this study, data from 96 Baluchi ewes genotyped using Illumina Ovine SNP50K BeadChip were used to identify genomic regions under selection associated with litter size in sheep. Phenotypic and pedigree data were collected at the Abbasabad Sheep Breeding Station. Based on records on different litters, ewes were divided into two groups: the case (two lambs per litter) and control (one lamb per litter).Quality control was conducted using the Plink software. The markers or individuals were excluded from the further study based on the following criteria: unknown chromosomal or physical location, call rate <0.95, missing genotype frequency >0.05, minor allele frequency (MAF) < 0.05, and a P-value for Hardy–Weinberg equilibrium test less than 10-6. To identify the signatures of selection, two statistical methods of FST and XP-EHH were used under FST and EHH software packages, respectively. We also calculated unbiased estimates of FST. Because the results were strongly correlated with the FST results, unbiased estimates have not been reported. In our study, all the SNPs ranking above 0.1 percentile of the distribution of test statistics were selected as candidates for the signature of selection. Gene ontology analysis for identified genes was performed using DAVID online database.

We used the FST and XP-EHH statistics to identify genomic regions that have been under positive selection associated with litter size in Baluchi sheep. Using FST approach, we identified 14 genomic regions on chromosomes 1 and 2 (two regions per chromosome), 3, 7, 9, 14, 18, 22, 23 (one region per chromosome), and X chromosome (three regions). Also, XP-EHH analysis identified nine genomic regions on chromosomes 2, 12, 13, and 22 (one region per chromosome), 7 (three regions), and X (two regions). Some of the genes located in identified regions under selection were associated with the number of lambs per lambing (ACVR1 and TGIF1), ovarian and follicle growth (DDX24), and fertility (FOXH1). Bone morphogenetic proteins are critical regulators of chondrogenesis during development which transduce their signals through three type I receptors namely BMPR1A, BMPR1B, and ACVR1/ALK2. TGIF1 is highly expressed in sheep ovaries suggesting that TGIF1 plays an important role in ewe reproduction. Also, TGF-β/SMAD signaling is critical in reproductive processes such as follicular activation, ovarian follicle development, and oocyte maturation. It has been evidenced that Ddx24 is highly expressed in sheep uterus affecting the development of ovaries and follicles. Foxh1 was first introduced as a transcriptional partner for Smad proteins and has been reported to play an important role in embryonic development. Results of gene ontology analysis identified two biological pathways namely defense response and cell motility which play an important role in the ovulation rate and the number of lambs per lambing. Reproductive activity and immune defenses can be mutually constraining, with increased reproductive activity limiting immune function and immune system activation leading to decreased reproductive function.

The results of this study identified candidate genes involved in the regulation of litter size in sheep suggesting that ACVR1 and TGIF1 genes can be considered as candidate genes related to the number of lambs per litter in sheep breeding programs to improve reproductive performance.

Gastrointestinal nematodes (GIN) represent a major health issue for livestock production systems worldwide. Haemonchus contortus is one of the most pathogenic GIN in small ruminants and causes serious losses to farmers, both in impaired production and in control with anthelmintics. It has long been recognized that differences in host resistance and susceptibility to parasitic infection exist in various sheep breeds and that genetics may play an important role in regulating host resistance, which has spurred efforts to control parasitic infection through selective breeding for naturally resistant sheep. A detailed understanding of the genes and mechanisms involved in expressing a resistant phenotype and the factors that regulate this response would facilitate the identification of candidate genes for selection. Recent advances in high-throughput technology, such as microarrays and RNA sequencing, have made a large number of transcriptome data accessible. As a result, researchers are now able to obtain more reliable results by integrating information from multiple sources. Accordingly, meta-analysis can be used as a useful and powerful tool to identify differential gene expression. It can also find out genes that their products are key molecules in response to the infection and use in animal breeding programs. The purpose of this study was to conduct a systematic review and meta-analysis on data collected from infected sheep with H. contortus and general analysis of changes in abomasal gene transcripts in response to infection using RNA-seq technology and bioinformatics tools. In this study, to identify infection-related genes, pathways, and molecular mechanisms underlying host resistance to this parasite, a meta-analysis was performed by combining two different datasets, including 70 samples of sheep H. contortus infection with Rankprod package of R software. After pre-processing, to remove heterogenicity across studies, batch effect correction was performed on gene expression data. The result of the principal component analysis showed that batch correction reduced the batch variation among the datasets. Meta-analysis was carried out and DEGs selected by meta-analysis were further analyzed and characterized. Enrichment analysis, as an efficient method for functional analysis of massive genetic data, was used to determine the biological process, molecular function, and cellular component of DEGs. Moreover, we searched upstream regions of DEGs for over-represented DNA motifs and functional analysis of discovered motifs. To explore the potential interaction network of the DEGs, the protein-protein interaction network among the DEGs was analyzed using the STRING database, which included direct and indirect associations of proteins. After analyzing the result derived from STRING analysis and expression change information for each DEG, the network figure was drawn for the selected DEGs (connected with one or more DEGs) by using the Cytoscape software and hub genes identified with the CytoHubba plugin of Cytoscape. Results derived from the meta-analysis showed a total of 1388 differentially expressed genes between resistant and susceptible sheep. Among them, 1137 were significantly upregulated, whereas 251 were downregulated across the datasets. In the identified DEGs, DEGs corresponding to ribosomal protein S3A, lysozyme C-1-like (LOC443320), and heterogeneous nuclear ribonucleoprotein K were the most strongly upregulated ones, while tenascin C and fibromodulin were the most strongly downregulated. Results from enrichment analysis showed these differentially expressed genes (DEGs) were involved in different biological processes such as one-carbon metabolism, translation, cell surface receptor signaling pathway, immune response, metabolic pathways, PPAR signaling pathway, etc. Moreover, searching in upstream regions of DEGs to find DNA motifs, were able to identify eight conserved sequence motifs. The functional analysis of these motifs revealed that they were involved in the positive regulation of gene expression, defense response, positive regulation of immune response, cellular calcium ion homeostasis, etc. Using the protein-protein interaction analysis also identified multiple hub genes such as albumin and CD4 which may show that improved immune response, induced by up-regulation different genes affects the creation of resistance. The mechanisms of sheep resistance to GIN infections involve complex immune responses. Our results offered overall insight into changes in the transcriptomes of resistant and susceptible sheep and molecular mechanisms of host resistance induced by H. contortus infection. We propose these DEGs as a useful resource of molecular biomarkers and potential candidate genes for breeding programs which can provide a basis for further research on this topic.

Quails are beneficial for their meat and eggs and have been the focus of poultry breeders in recent decades. Egg quality traits are usually divided into external (such as shell quality and egg weight) and internal (like yolk and albumen parameters) traits. Shell characteristics are important factors to determine egg quality. The breakage of quail eggs can be occurred in various processes such as collection, packaging, and transportation or in the setter and hatcher devices, and leads to serious economic problems for the breeders in the poultry industry. The size and weight of the quail egg are important factors in the marketability of this product. From an economic point of view, the yolk is the most valuable part of the egg. The yolk is a concentrated source of nutrients, as well as it has a significant effect on the weight of the chicken. The ratio of yolk to white in quail eggs has been reported to be 35 to 65, which is higher in comparison to laying hens. Egg quality can be affected by different factors such as genotype, breed, strain, age, management, nutrition, temperature, and illness. Although there have been many studies to determine the environmental factors on traits related to egg quality in quail, there are few studies to investigate the effect of genetic factors and different mating systems on the occurrence of these traits. Therefore, the present study was conducted to estimate genetic parameters of external and internal egg quality traits in wild and white strains of Japanese quails.

A total number of 570 laying quails were obtained from pure and cross-mating groups of wild and white Japanese quails. The base population was divided into four mating groups including two pure and two crossed mating groups (group 1: wild male × wild female; group 2: white male × wild female; group 3: wild male × white female, and group 4: white male × white female). The studied traits included external (egg weight, length, and width, shell weight and thickness, and egg shape index) and internal (yolk weight, albumen weight, yolk index, albumen index, and internal egg quality unit) egg quality traits. The effects of hatch and mating groups on traits related to egg quality were investigated. The means of traits related to external and internal egg quality were compared using the Duncan test. (Co)Variance components and genetic parameters of traits related to external and internal quality traits were estimated by multiple trait animal models and restricted maximum likelihood using ASREML software.

The effect of the hatch on egg weight, shell weight, and albumen weight was significant (P<0.05) and the eggs from the second and third hatches showed higher egg weight, shell weight, and albumen weight in comparison to the first hatch (P<0.05). The difference between mating groups was not significant (P>0.05) except for albumen and egg weights. The laying quails from the pure white mating group showed lower albumen and egg weights (P<0.05). The heritability estimates for traits related to external egg quality varied from 0.16 (egg shape index) to 0.39 (egg weight). These estimates for traits related to internal egg quality ranged from 0.10 (internal quality unit) to 0.31 (yolk weight). Genetic correlations were estimated from -0.33 (between egg length and egg shape index) to 0.71 (between egg length and width) for traits related to external egg quality, and from -0.37 (between yolk weight and yolk index) to 0.71 (between albumen weight and albumen height) for traits related to internal egg quality, respectively. Phenotypic correlations varied from -0.32 (between egg length and egg shape index) to 0.61 (between egg length and width) for traits related to external egg quality, and from 0.11 (between yolk index and albumen height) to 0.59 (between the internal quality unit and albumen height) for traits related to internal egg quality, respectively.

Heritability estimates for traits related to egg quality were moderate which indicates the significant effect of additive genes on the occurrence of these traits. Positive genetic correlations between egg weight and traits related to external egg quality, as well as appropriate genetic correlations between traits related to internal egg quality showed that selection based on some egg traits like egg weight and internal egg unit can effectively lead to the improvement of other egg quality traits.

Honey bees need nutrients such as protein, carbohydrates, lipids, and minerals. Among nutrients, protein is of particular importance due to its role in brood rearing, developing hypopharyngeal glands and ovaries, longevity, bee immunity, bee weight, and flight muscle development. The protein stored in the body of bees is vitally important in strengthening the immune system, producing royal jelly, and especially in wintering. The honey bee obtains its protein needs through the digestion of different pollen grains with different amounts of protein in the midgut. Among the amino acids required by honeybees, the most important are leucine, isoleucine, and valine. Pollen quality depends on the supply of amino acids required by honey bees. Most pollen grains are poor and deficient in isoleucine. This study aimed to evaluate the effect of different levels of isoleucine on hemolymph protein and the growth of hypopharyngeal glands in worker bees
To investigate the effect of different levels of isoleucine on hemolymph protein and the growth of hypopharyngeal glands in worker bees, an experiment was conducted in a completely randomized design with five experimental diets and four replications (cages). 100 honey bees were kept in each cage. Diets included 1 M sucrose syrup (control), and different levels of isoleucine containing 0.5, 1, 1.5, and 2 micrograms per liter of 1 M sucrose syrup. Before starting the experiment, the egg frames in the hives were marked so that the bees were of the same age at the time of the experiment. The cages were placed in an incubator at a temperature of 34°C and a humidity of 60%. Samples were taken to measure hemolymph protein on days 1, 6, 12, and 18 of 10 bees and for growth of hypopharyngeal glands on days 3, 6, 9, 12, and 15 of three bees. The hemolymph protein amount was measured by UV spectrophotometer and Bradford method and the growth of hypopharyngeal glands by the diameter of the ascites.
The results showed that the difference in hemolymph protein and the growth of hypopharyngeal glands of bees was significant in the whole experiment period and at different ages (P<0.01). A comparison of the experimental treatments revealed that the 1.5 μg isoleucine treatment had the highest amount of hemolymph protein among the diets (6.65 μl/μg), and the difference with other treatments was significant (P<0.01). The control treatment had significantly the lowest hemolymph protein among the experimental diets (3.57 μl/μg). The use of high amounts of isoleucine decreased the hemolymph protein. Therefore, the appropriate level of isoleucine in a honey bee diet is 1.5 μg. In all diets, except the control diet, the highest amount of hemolymph protein was observed on the sixth day, but in diet control, the amount of hemolymph protein decreased. Regarding the hypopharyngeal glands, the comparison of the treatments revealed that the 1.5 μg isoleucine treatment had the largest acini diameter (0.083 mm) and its difference with other treatments was significant (P<0.05). The control treatment (0.065 mm) significantly had the lowest acini diameter among the experimental treatments. Comparing the diameter of acini at different ages showed that the largest diameter of acini was observed at three days of age and the smallest diameter was seen at 15 days of age in all treatments. Some of the increase in hemolymph protein is likely due to the consumption of isoleucine, and some of it is due to the increase in the activity of hypopharyngeal glands because the consumption of isoleucine has increased the growth of hypopharyngeal glands.
In this experiment, it was found that the hypopharyngeal glands grew to the maximum level, and then, the amount of protein in the hemolymph of bees reached the highest level. Based on the findings of this research, the use of isoleucine in the diet of bees can increase the production of royal jelly and brood rearing due to its influence on the growth of hypopharyngeal glands and the amount of hemolymph protein. The appropriate level of this amino acid in bee nutrition is 1.5 μg per liter of sucrose syrup because consuming more than this optimum level harms hemolymph protein and the growth of hypopharyngeal glands.The difference between hemolymph protein and hypopharyngeal gland growth of bees was significant in the whole experimental period and at different ages (P <0.01). Diets containing 1.5 μg of isoleucine and control diet had the highest (6.65 μl / μg) and the lowest (3.57 μl / μg) of hemolymph protein and the highest (0.083 mm) and lowest (0.65 mm), respectively. There was no significant difference between hemolymph protein in syrup containing 0.5 and 2 micrograms of isoleucine and growth of hypopharyngeal glands in treatments of 0.5, 1 and 2 micrograms of isoleucine. Therefore, the best level of isoleucine used in bees for proper production performance was the feeding of syrup containing 1.5 μg of isoleucine.