فصلنامه تحقیقات تولیدات دامی
سال سیزدهم شماره 2 (تابستان 1403)

The CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated Protein9) method can create a nucleotide sequence complementary to the target sequence in the desired gene by Guide-RNA (gRNA) together with the Cas9 protein, which is a cutting enzyme for cutting in the both DNA strands of the desired sequence accurately and clearly. The DAZL (Deleted in azoospermia-like) gene encodes potential RNA-binding proteins that are expressed in male and female germ cells before and after birth. DAZL, which acts by post-transcriptionally binding mRNA in 3' untranslated regions, regulates the germ cell cycle. DAZL initiates the sexual differentiation of embryonic germ cells. The transfer and transplantation of gene-edited germ cells into recipient males is an effective method for targeted mutagenesis engineering. In mice knocked out for the Dazl gene, the number of testicular stem cells was reduced and it was found that the DAZL gene plays an important role in the differentiation of spermatogonial cells. The purpose of the current research is to edit the DAZL gene by knocking it out using the CRISPR/Cas9 technique and transferring the somatic cell nucleus into the genome of the Bakhtiari goat embryo. The inactivation of the gene will be investigated both at the level of the embryonic cell and the resulting embryo. There are no reports on the production of Bakhtiari goat cells and embryos edited for the DAZL gene by CRISPR technique and somatic nuclear transfer to improve any traits, including reproductive ones. To target the DAZL gene and explore (predict) potential off-target genomic sites, guide RNA (20 bp sequences) immediately upstream of each 5′-NGG in the DAZL gene was designed using the CHOOPCHOOP tool. Plasmid pX459 (9151 bp) was used to insert the gRNA sequence into the CAS9 vector and determine the characteristics and replication of the vector. This plasmid encodes the Cas9 protein along with the puromycin resistance gene under the promoter/enhancer, CAGGS, as well as the gRNA scaffold under the U6 promoter. Plasmid pX459 was digested by BbsI-HF (NEB #R3539) at 37°C for 10 min, followed by purification by NucleoSpin Gel and PCR Clean-up Midi kit (#740,986.20, Machery/Nagel). The purified piece was kept at -20°C for later use. Ligation of oligoduplex carrying diluted gRNA sequence (1:20 ratio from 10 μM source) (1 μL), digested pX459 vector (50 ng), 10x T4DNA ligase buffer (2 μL), and T4DNA ligase (1 μL) in the final concentration of 20 μL of the reaction was carried out. Ligation mixture transformation was performed with NEB 5-alpha Competent E. coli (#C2987I) and then placed on agar culture medium with 100 μg/mL ampicillin and incubated at 37°C overnight. From the cultured plate, five colonies were selected and each was cultured in LB culture medium, followed by miniprep plasmid extraction (Genejet Plasmid miniPrep kit, #K0502). Plasmids carrying the CRISPR system were transferred into cells through an electroporation system. The present results proved the possibility of knocking out the DZAL gene using the CRISPR/Cas9 technique in both fibroblast cell lines and Bakhtiari goat embryos. We performed somatic cell nuclear transfer (SCNT) to examine the embryonic developmental capacity to transition to the cleavage and blastocyst stages. The embryos created by knocking out the DAZL gene could grow and develop. The sequencing analysis using DECODR (Deconvolution of Complex DNA Repair) software showed the knock-out rate of the DAZL gene in fibroblast cells was 83.3% and in cloned blastocysts, it was 55.3%. The rate of blastocyst formation resulting from the cloning process with knockout cells was not significantly different from the control group. Based on these studies, we decided to edit the DAZL gene to produce knockout goat embryos by SCNT. The embryos were examined and evaluated with the help of PCR-RFLP test and sequencing. It turned out that in the first iteration, a knockout of 60.66% (the first ten embryos were paid), and in the second iteration, a knockout of 49.99% (the second ten embryos were paid) were achieved. Such mutations have not been described or detected in Iranian Bakhtiari goats, although polymorphisms have been identified. Using genetic sequencing, the mutations were of the INDEL type and were all in the form of frameshift, which resulted in a change in the protein. The CRISPR/Cas9 system easily influences the desired gene and can be used as a strategy to produce livestock animals that are superior in milk and meat production, reproduction, quality, disease resistance, etc. This research demonstrated the possibility of gene editing using the CRISPR/Cas9 technique in fibroblast cell lines and Bakhtiari goat embryos. The blastocytes produced can be used for transfer to the recipient animal and production of DAZL gene knockout goats for further study and optimization of spermatogonial stem cell transplantation.

The racing homer pigeon is one of the most popular ecotypes that have a remarkable navigation ability, compared to the Rock dove pigeon, which has an undesirable navigation ability. Therefore, in this research, these two ecotypes were used for comparative genomics and identification of genetic variants associated with navigation traits and flight ability. Functional examination of related variants and genes can help identify putative genes associated with navigation mechanisms and elaboration of the relevant biological pathways. On the other hand, these pigeons can be used as an animal model to investigate biological processes related to memory and learning ability also identifying key genes and biological processes related to memory and learning processes. This study aimed to identify the genetic variants related to the navigation ability in two ecotypes of pigeons.

Transcriptome data (eight samples) related to the hippocampus of Rock dove pigeons (weak routing ability) and Racing homer (good routing and long-distance homing ability) were downloaded from the EBI database with the accession number PRJNA532674. The reference genome and all available annotations related to pigeons were downloaded from the Ensembl database (ftp://ftp.ensembl.org). Data quality control was performed based on read length, GC content, unread bases, duplicate reads, and Phred index. To identify the variants, the fixed ploidy algorithm, and the following parameters were used. The score of the base variant was 30, the nighber base score of the variant was 20, and the frequency of the base variant was set at 30%. Quality control of reads, mapping, and identification of different genetic variants between Rock and Homing groups were performed with CLC Genomics workbench software. The functional analysis of different variants was performed by gene ontology analysis by the VEP tool in the Ensembl database with a significance level of 5%. Gene ontology analysis was performed at three levels: molecular function (MF), biological processes (BP), and cellular components (CC).

Around two million genetic variants, were identified for the eight samples (944325 for Racing homer and 1055277 for Rock dove pigeon), of which 2800 different genetic variants were reported between the two groups. The variants were classified into four categories including single nucleotide polymorphisms (71.1%), multiple nucleotide variants (16.8%), insertions (6.5%), and short deletions (5.5%) for Racing homer pigeons. The frequencies of corresponding variants for the Rock dove pigeon were 73.2%, 12.5%, 7.5%, and 68%, respectively. All reads showed an alignment of above 90% with the reference genome which indicated a good alignment. The results of the gene ontology analysis showed that different variants are related to the LDHA gene. This gene is important in the flight power and anaerobic metabolism of cells by coding the lactate dehydrogenase A enzyme. Birds with a mutated version of the LDHA gene perform better in competitions for flying power and navigation. Another finding of this study was the association of genetic variants with the DRD4 gene, which is important in controlling behavioral patterns, memory, and learning. Protein metabolism pathways were also one of the important results of this research, due to their essential role in the differentiation of nervous system cells. For example, the Ubiquitin protein acts as a messenger for proteases by marking other proteins and facilitates the digestion of ineffective proteins in the cell. Also, this protein has a regulatory role in the activity of other proteins. One of the results of gene ontology analysis at the molecular function level was related to protein Serine/threonine kinase activity, which confirms the results of biological process analysis. Also, the PIK3C2A candidate gene was suggested for this molecular effector. This gene, by encoding Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit alpha, plays a fundamental role in regulating cell activity such as the transfer of glucose to plasma, the secretion of insulin and the response to UV or other stresses.

The results of this study confirmed that the homing pigeon and rock pigeon ecotypes had different genetic potential in terms of navigation ability. Differences in routing ability between two studied ecotypes should be related to mutations and changes in genomic regions during evolution. However, the identified genetic variants must be confirmed under laboratory conditions using PCR or other techniques. The identified variants can be used as a model for further research into human diseases and the identification of paralog genes related to human memory and learning ability.

 In dairy cows, milk yield changes over the lactation period. Lactation curve modelling could be of importance from nutritional management and genetic selection point of view. The trajectory shape of the lactation curve has inclining and declining slopes which determine total amount of milk yield during the lactation. A great number of linear and nonlinear mathematical models have been so far utilized to describe the shape of the lactation curve in dairy cows. Among the models, mechanistic functions are considered to be more accurate than empirical models in terms of taking account of biological mechanisms undertaken in mammary gland to produce milk. To our knowledge, no research has been carried out to use a complex mechanistic model for describing the lactation curve of Iranian dairy cows. Based on this, the present research aimed to apply a five-parameter nonlinear mechanistic model for mathematical description of the shape of the lactation curve in Iranian first-parity dairy cows.

Animal Breeding Centre of Iran provided the data used in this research. Initial data in separate files were merged and edited by the SPSS software. Final data comprised 5596039 milk test day records from 821153 first-parity cows distributed in 579 herds of 26 provinces over the country. The cows were the progeny of 7957 sires and 530394 dams and calved between 1996 and 2020. The cows were categorized into two groups based on the age of first calving: <=25 or >25 months. Also, two groups of cows were created based on the proportion of Holstein gene inheritance: <100% (grade cows) or 100% (pure Holstein cows). A five-parameter nonlinear mechanistic model was applied to mathematically describe the shape of the lactation curve. The fitted mechanistic model had five parameters including MSmax (maximum milk secretion potential of the lactation), GR (relative proliferation rate of secretory cell number during early lactation), MSLmax (maximum secretion loss), NOD (proportion of parenchyma cell dead at parturition), and DR (relative decline rate in cell number) which their estimates were obtained for different seasons and ages of calving, as well as different genotypes (grade and pure Holstein) using NLIN procedure of the SAS software. Based on the estimated parameters of the model in each year of calving, phenotypic trends were calculated using the SPSS software.

The results indicated that Iranian first-parity dairy cows reach to peak of milk production during the third month of the lactation curve. Based on standard deviation as well as coefficient of variation (CV) of milk test day milk records, maximum variation was observed in the last month of the lactation period. The findings also indicated that cows calved in autumn had the greatest MSmax and GR while cows calved in spring and summer had the lowest magnitude for these parameters (P<0.05). The minimum magnitude of MSLmax and NOD were detected for the cows calved in summer (P<0.05). Regarding the DR parameter of the model, cows calved in spring were observed to have a minimum value. Maximum MSmax, Gr, and DR values ​​were determined for cows calving up to 25 months of age, while maximum MSLmax and NOD values ​​were observed for cows of later ages (P<0.05). Compared to pure Holstein cows, grade cows had higher MSLmax and NOD values, but other parameters were found to be greater in the pure Holstein cows (P<0.05), suggesting that the maximum milk secretion potential during lactation in pure Holstein cows is expected to be higher than that of grade cows. Annual phenotypic change trends were found to be 0.376 kg/year (R2=0.9), 0.00009869 (R2=071), -0.146 kg/year (R2=0.59), -0.011 (R2=0.85), and 0.001 (R2=0.85) for MSmax, GR, MSLmax, NOD, and DR, respectively (P<0.0001).     

This study found that the parameters of the mechanistic model fitted to the milk test day records of Iranian primiparous dairy cows are significantly influenced by the age and season of calving as well as the genotype of the cow. Cows calving in summer are expected to have more persistency. Moreover, pure Holsteins are more persistent than grade cows. Positive and negative annual trends have been detected for MSmax and MSLmax parameters, respectively, during 1996-2020 indicating a favorable increase in maximum milk secretion potential of the lactation and also a favorable decrease in maximum secretion loss over that period.

Molecular markers that reveal polymorphisms at the DNA level now play a key role in animal genetics. However, the selection of molecular markers is crucial depending on the purpose, viz. this depends on different molecular biology techniques and their effects. Over the last decade, interest in identifying genes or genomic regions targeted by selection has grown. Identifying selection signatures can provide valuable insights into the genes or genomic regions that are or have been under selection pressure, which in turn leads to a better understanding of genotype-phenotype relationships. Type characteristics are important for breed identification and classification and are also positively correlated with body weight. This study aimed to identify effective genes and genomic regions under positive selection signatures in different goat breeds using selection signature methods. For this purpose, FST and hapFLK analyses were performed using the genome-wide single nucleotide polymorphisms (SNPs).
In this research, the information from 728 goats of four different breeds was used to identify genomic regions associated with type traits. To determine the genotype of the samples, Illumina caprine Bead Chip 50K was used. The genomic information of goat breeds was extracted from the Figshare database. Quality control was performed using the Plink software. The markers or individuals were excluded from 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 10-3. After quality control, 36861 SNPs from goat SNP chip 50K from 691 goats remained for further analysis. To identify the signatures of selection, two statistical methods of FST and hapFLK were used under the software packages FST and hapFLK, respectively. Candidate genes were identified using the Plink v1.9 software and the Illumina gene list in R by SNPs located in the highest  FST and hapFLK values. In addition, the latest published version of the animal genome database was used to define QTLs associated with economically important traits at identified loci. The GeneCards (http://www.genecards.org) and UniProtKB (http://www.uniprot.org) databases were also used to interpret the function of the obtained genes.
The FST and hapFLK statistics were used to identify genomic regions subjected to positive selection associated with type traits in four goat breeds. Using the FST approach, we identified eight genomic regions on chromosomes 3, 4, 7, 13, 15, 18, 20, and 29. The identified candidate genes associated with type traits in these genomic regions included TGFBR3, CALCR, ACAD8, BCAR1, and ADAMTS6. Some of the genes located in the identified selection regions were directly and indirectly related to cell differentiation and proliferation, skeletal muscle growth and development, body length, calcium channel regulation, muscle fiber homeostasis, protein synthesis, and muscle cell size. Some of these genes in the selected regions were consistent with previous studies. The results of the reported QTLs in the selected regions and the bovine orthologous regions were QTLs located in the identified regions that were related to average daily gain, body weight, trunk width, and metabolic body weight. Furthermore, the results of the hapFLK statistics in this research led to the identification of five genomic regions on chromosomes 1, 5, 6, 13, and 30, and they were in the 99.9th percentile of all hapFLK values. The identified candidate genes associated with the type trait in these genomic regions included FNDC3B, STAB2, and CCNY. They were found to have different functions in fibroblast proliferation and bone cell differentiation.
Various/different genes that emerged in studied regions can be considered candidates for selection based on their function. By the way, various genes found in these regions can be considered candidates for selection based on their function. Most of the selected genes were found to be consistent with some previous studies and to be involved in production traits. A survey of extracted QTLs also found that these QTLs are involved in some economically important traits in goats, such as average daily gain and body weight in yearlings. However, further association and functional studies are required to demonstrate the importance of the genes obtained from association analyses. Leveraging these findings can accelerate genetic progress in breeding programs and help understand the genetic mechanism that controls these traits.
In this research, to identify genomic regions under selection associated with type traits were used the information obtained from 728 goats of different breeds including Beetal, Daira Deen Panah, Barbari, Teddi, In order to determine the genotype of the samples, Illumina caprine Bead Chip 50K were used. The genomic information of goat breeds was extracted from the figshare database. 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 10-3. After quality control, 36,861 SNPs from Goat SNP chip 50K on 691 goats were remained for the future analysis. To identify the signatures of selection, two statistical methods of FST and hapFLK were used under FST and hapFLK software packages, respectively. Candidate genes were identified by SNPs located at 1% upper range of FST and hapFLK using Plink v1.9 software and the gene list of Illumina in R. Additionally, the latest published version of Animal genome database was used for defining QTLs associated with economic important traits in identified locations. GeneCards (http://www.genecards.org) and UniProtKB (http://www.uniprot.org) databases were also used to interpret the function of the obtained genes. We used the FST and hapFLK statistics to identify genomic regions that have been under positive selection associated with type traits in four goat breeds. Using FST approach, we identified eight genomic regions on chromosomes 3, 4, 7, 13, 15, 18, 20, and 29 chromosome. The identified candidate genes associated with type trait in these genomic regions included TGFBR3, CALCR, ACAD8, BCAR1, ADAMTS6. Some of the genes located in identified regions under selection were associated with the cell differentiation and proliferation, skeletal muscle growth and development, body length, calcium channel regulation, muscle fiber homeostasis, protein synthesis and muscle cell size which can be directly and indirectly related to the trait of the type traits. Some of these genes in the selected regions were consistent with previous studies. Result of the reported QTLs in the selected regions and the orthologous regions of cattle were located in the identified regions, QTLs related to average daily gain, body weight, rump width and body metabolic weight. Also, the results of hapFLK statistics in this research led to the identification of five genomic regions on chromosomes 1, 5, 6, 13, and 30, and they were in the 99.9 percentile of all hapFLK values. The identified candidate genes associated with the type trait in these genomic regions included FNDC3B, STAB2 and CCNY. It was determined that they had different functions in proliferation of fibroblasts and differentiation of bone cells. Result of the reported QTLs in the selected regions and orthologous cattle in the identified regions, QTLs related to metabolic body weight were located. various genes that were founded within these regions can be considered as candidates under selection based on function. Most of the genes under selection were found are consistent with some previous studies and to be involved in production traits. Also, survey on extracted QTLs was shown that these QTLs involved in some economical important traits in goat such as average daily gain and body weight in yearling. However, it will be necessary to carry out more association and functional studies to demonstrate the implication of these genes. However, it will be necessary to carry out more association and functional studies to demonstrate the implication of these genes and survey on QTLs related to selected regions. However, will be necessary to carry out more association and functional studies to demonstrate the implication of genes obtained from association analyses. Using these findings can accelerate the genetic progress in the breeding programs and can be used to understand the genetic mechanism controlling this trait.

Colostrum is a secretion product produced by the mammary glands immediately after birth. This vital and unique composition is designed to meet all the animal's needs when it cannot eat. For various reasons, maternal accidents can occur during pregnancy and birth, making it impossible to produce appropriate colostrum for the newborn calf. Therefore, the main task of the farmer is to provide suitable replacement colostrum as quickly as possible so that he can maintain the results of his year-long efforts to continue the generation. Various methods are used to preserve colostrum. Methods of colostrum storage include fermentation and freezing. Fermentation of the colostrum results in physical and chemical changes in the colostrum that can aid in the transfer of nutrients to the calf. This research was carried out to study and compare the effect of different methods of colostrum preservation on the efficiency of newborn calves of the Simmental breed and to recommend the use of one of the more suitable methods so that if the farmer does not have access to suitable colostrum, he can use.
To carry out this study, 32 newborn Simmental calves with an average weight of 39.5 ± 3.2 kg were used. The calves were divided into four equal groups (eight replicates per treatment), including 1. group fed with fresh colostrum from the mother (control), 2. group fed with fermented colostrum without any additives (fermented without additives), 3. the group that was fed colostrum fermented with low-fat yogurt (fermented with yogurt), and 4. the group was fed with colostrum that was kept frozen (frozen) in the freezer and heated to a temperature of 37 °C before consumption. Initially, a colostrum bank was used with 110 liters of colostrum from the first and second lactation cows, which started colostrum production at the same time by synchronizing parturition. Colostrum samples were separated and prepared in polyethylene (PET) plastic containers with a volume of two liters. In the fermented colostrum group, after filling the containers of the group with yogurt, two percent yogurt was added, and the containers were completely sealed and stored at room temperature until the experiment. The dishes belonging to the frozen group were stored in the freezer at -20 °C. In the first two days, all calves received two liters of colostrum per meal twice daily in the morning and evening. Feed was consumed daily. Calves were weighed at the beginning of parturition, on the 30th and 60th days of the schedule in the morning and before feed distribution, and from the 27th day of the calves' birth, the apparent digestibility of nutrients (dry matter) was measured. Samples were taken from three-day experimental rations and feces. Blood was collected from the jugular vein on days 0, 1, and 30. After serum separation, blood factors such as glucose, cholesterol, triglyceride, and total blood protein were measured. At the beginning of the course and the end of the project, body parameters were measured to check the growth status of the calf: hip width, height from withers, chest circumference, body length, chest depth, wrist circumference, and eye relief.
The results of different treatments of colostrum consumption in newborn calves concerning blood biochemical parameters showed that the amount of glucose in all three sampling times was not significantly different in all treatments, and the amount of blood protein and triglycerides were also not affected by the treatments (P>0.05). Colostrum consumption with different storage methods had no significant influence on the amount of concentrate consumed, the digestibility of the dry matter consumed, the final weight of the calf, the daily weight gain, and the feed conversion ratio (P>0.05). Also, the results showed that there were no significant changes in physical indices among different groups (P>0.05). The use of colostrum with different storage methods had no difference in the amount of cells associated with the immune system, including types of white blood cells, and therefore the immune conditions were similar in all groups (P>0.05).
The results of this study showed that consumption of colostrum in simply fermented form or with the addition of fat-free yogurt, as well as colostrum stored in frozen form, had a positive influence on blood parameters in newborn calves compared to the control group that used fresh maternal colostrum. The use of colostrum stored in any of the mentioned methods, depending on the conditions of animal husbandry, can help provide the colostrum needed by the calf and solve the problem of the animal breeder.

 Suboptimal levels of kid survival are the largest contributor to reproductive wastage in goat flocks. This results in substantial loss of production, producer, and industry income, and is increasingly being perceived as poor animal welfare. Improving kid survival is therefore a priority for the industry. Nutrient provision during gestation not only affects maternal status and reproductive performance but also affects prenatal and postnatal offspring growth and health. Although trace minerals (TM) are needed by the body in small amounts, they are essential nutrients for several metabolic functions such as growth, development, reproduction, and immunity. Furthermore, newborn animals are dependent upon their dams for the transfer of these nutrients via the placenta and the mammary gland. The antibodies obtained from colostrum are the only defense mechanism against environmental factors in neonatal ruminants. Inadequate nutrition of the dam, immune system suppression, and stress factors may lead to the production of low-quality colostrum. Management and feeding of high-quality colostrum can reduce kid mortality, strengthen immunity, and increase animal life span. Nutrition affects the development of the mammary gland, the onset of lactogenesis, and colostrum production, either by affecting some of the hormones that control these processes or by contributing nutrients that are in demand at this stage of pregnancy. Selenium plays an important role in preventing impaired function of the immune response. Copper deficiency has been shown to result in lowered bactericidal activities of blood leukocytes in ruminant animals. Zinc sufficiency has also been linked to proper immune functions. Therefore, this study aimed to investigate the effect of TM injection in late pregnancy on colostrum quality and plasma metabolites of Lori does and their kids.
Thirty Lori mature does with an average body weight of 40 kg and an age of 2-3 years were used. One month before the expected kidding, animals were divided into two groups (n=15 does/group) and randomly assigned to experimental treatments. Experimental treatments were no injection of trace minerals (Control; C) and injection of 1 mL of TM at four and two weeks before expected kidding. Blood samples were taken through the jugular vein. Each mL of TM solution contained 2.5 mg of Cu, 1.25 mg of Se, 5 mg of Mn, and 5 mg of Zn. All does were kept in similar nutritional and managerial conditions from mating to one month before kidding.
Results showed that plasma concentrations of glucose and total cholesterol (TC) tended to be higher and lower in the TM group at day 7 before kidding, respectively (P=0.06). Experimental treatments did not affect plasma triglyceride (TG), Ca, and Mg concentrations, and glutathione peroxidase (GPX) activity of does at day 7 before kidding (P>0.05). Whereas, TM injection before mating decreased plasma malondialdehyde (MDA) and increased total protein (TP), BRIX index (BI), superoxide dismutase (SOD), and total antioxidant activity at day 7 before kidding (P<0.05). Colostrum of does received TM had a higher fat, protein, and BI content and a lower lactose content than the colostrum of the C group (P<0.05). Plasma concentrations of glucose and MDA were lower in kids born from does received TM than those born from the C group (P<0.05). Experimental treatments did not affect plasma concentrations of TC, Ca, and Mg concentrations in kids (P>0.05). Kids born from does received TM had higher plasma concentrations of TG, TP, BI and SOD, GPX, and total antioxidant activity than those of the C group (P<0.05).
According to the results of the present experiment, injection of a TM solution containing Cu, Zn, Se, and Mn four and two weeks before birth increased the concentrations of TP and BI as well as the activity of antioxidant enzymes (SOD, GPX) and antioxidant capacity and reduced MDA concentration in Lori goat plasma. This improved the quality of colostrum produced by these does. Kids born from does receiving trace minerals had higher plasma concentrations of TP, BI, and activity of antioxidant enzymes (SOD, GPX), higher total antioxidant capacity, and lower concentrations of MDA compared to the C group. Therefore, this strategy may have beneficial effects on the health, viability, and performance of kids before weaning.

Honey bees need nutrients such as protein, carbohydrates, lipids, minerals, and vitamins. Among nutrients, vitamins are particularly important for their roles in brood rearing, hypopharyngeal gland development, ovary development, longevity, bee immunity, bee weight, and flight muscle development. The vitamins stored in the body of the bees are vitally important in strengthening the immune system, normal growth, and development of broods, producing royal jelly, longevity of bees, and especially wintering quality of honey bees. Among the vitamins required by the honey bees, the most important are thiamine and riboflavin. Pollen quality depends on the supply of vitamins required by honey bees. Most pollen grains are poor and deficient in thiamine and riboflavin. This study aimed to investigate the effect of thiamine and riboflavin on population growth, functional traits, and fat and protein reserves of the bee body in Iranian honey bee colonies.

An experiment was conducted in a completely randomized design with four treatments and seven repetitions from April 2022 to September 2023 in the climatic conditions of Kurdistan province, Iran. Experimental treatments included the sugar syrup (control), thiamine (1500 ppm), riboflavin (1500 ppm), and the combination of thiamine and riboflavin (1500 ppm). From the beginning of 14 April 2023, experimental treatments were fed with the mentioned concentrations of vitamins thiamine, riboflavin, and their combination of 0.5 liters every other day for 45 days and 15 days without feeding. Then, traits such as honey production, pollen collection, population (adults and broods), and protein and fat content of carcass were measured in experimental treatments.

The results of the effect of thiamine, riboflavin, and their combination on the population of adult bees showed that the addition of thiamine and riboflavin in the bees' nutrition was significantly effective in the increase of adult bees' population in the investigated periods as well as the average of the entire period (P<0.05). A comparison of the averages of the experimental treatments revealed that the highest and lowest adult bee populations were associated with the treatments fed the vitamin combination and the control group, respectively. The results of the effect of thiamine, riboflavin, and their combination on the population of broods showed that the addition of thiamine and riboflavin in the diet of bee colonies during the studied periods did not have a significant effect on the increase in the population of newborns, but it showed a significant effect on the average of the entire period (P<0.05). The results of the mean comparison showed that the highest population of broods in the studied honey bee colonies was related to the treatments fed with the combination of thiamine and riboflavin, and the lowest population of broods was related to the control treatment. The results of the effect of thiamine and riboflavin on honey and pollen production traits showed that the use of vitamins and their combination in honey bee nutrition caused a significant increase in honey and pollen production traits of experimental treatments (P<0.05). The results of the mean comparison showed that the highest and lowest amounts of honey and pollen production of the studied honey bee colonies were observed in the treatment fed with the combination of vitamins and the control group, respectively. The effect of thiamine and riboflavin on the carcass protein and fat showed that the use of thiamine, riboflavin, and their combination in feeding the colonies had a significant effect on the amount of carcass protein and fat (P<0.01). A comparison of treatments showed that honey bees in the treatment fed the vitamin combination had the highest carcass protein and fat, and honey bees in the control treatment had the lowest carcass fat and protein.

Based on the results of the present experiment, it could be concluded that the use of thiamine and riboflavin, and their combination in feeding honey bees had a positive effect on population growth, functional traits, and fat and protein reserves of the bees' body in the colonies and improved the performance and increased the economic efficiency of the colony.