Complexes were prepared by the physical mixing (PM) and solvent evaporation (SE) methods. The prepared inclusion complexes were meticulously characterized by X-ray diffractometry (XRD), scanning electron microscopy (SEM), and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra. To verify our findings, the inclusion complexes were evaluated by equilibrium solubility, in vitro drug release profile, kinetic models, and antidiabetic and antioxidant activities in animal models. Our results demonstrated that the solubility and drug release profile were found to be enhanced through binary as well as ternary complexes. Notably, ternary complexes with a ratio of 1 : 9 : 1 showed the highest solubility and drug release profile compared to all other preparations. Data on antioxidant activity indicated that the ternary complex had the higher total antioxidant status (TAS), superoxide dismutase (SOD), and catalase (CAT) activity than the binary complex and Glc alone, in contrast to the diabetic group. In vivo antidiabetic activity data revealed a high percentage reduction in the blood glucose level by ternary complexes (49–52%) compared to the binary complexes . HPβCD and Tryp provide a new platform for overcoming the challenges associated with poorly soluble Glc by providing greater complexing and solubilizing capabilities and imparting ancillary benefits to improve the drug's antidiabetic and antioxidant activities.The poor solubility of the antidiabetic drug gliclazide (Glc) is due to its hydrophobic nature. This research is aimed at improving Glc's solubility and drug release profile, as well as at investigating additional benefits such as bioactivity and antioxidant activity, by forming binary complexes with HP Scientists theorize that this disorder amplifies oxidative stress levels due to depletion in ascorbic acid . Glc 1- 2], a B, a B2], in vitro and in animal models. When compared to other sulfonylurea drugs, Glc has a higher potential for free radical scavenging because the scavenging part of the molecule is an aminoazabicyclo-octane ring, which is not evident in other sulfonylurea drugs [μg/mL within 2–8 hours. Regular Glc administration raises Cmax and tmax levels, and after 48 hours of 40–120 mg doses, a steady-state concentration is achieved. Because of its high affinity for protein binding (85–97%), a small volume of Glc (13 to 24 L) is distributed in both healthy volunteers and patients. Glc half-life (t1/2) is approximately 8.1 to 20.5 hrs, and plasma clearance is 13 mL/min (0.78 L/hr). Because the drug is easily metabolized and excreted in the urine, renal insufficiencies have no effect on Glc's pharmacokinetic properties [Glc has recently received considerable attention for revealing other pharmacological properties like antioxidant and anti-inflammatory activities ea drugs . Accordiea drugs , peak plding 85–9%, a smalin situ micronization, nanospheres, solid complexes, and prodrug formation [Glc's poor solubility due to its hydrophobic nature is a major flaw in formulation, making it challenging for scientists to produce dosage forms that may improve its solubility and bioavailability . Poor soormation . Recent ormation , 12 coulormation , 14 usinormation . In addiormation , 17.βCD is demonstrated as a type of cyclodextrin with seven glucose units and hydroxypropyl as a substituent 7(C3H7O)45, an average molecular weight of 1396 (anhydrated), and a substitute of 0.67 hydroxypropyl groups per glucose unit, highly soluble in water 45 g/100 mL. The literature shows that cyclodextrin complexes boost and maintain the antioxidant activity of drugs, providing an additional benefit [New technologies and raw materials assisted in developing stable, safe, and efficient drug carriers with fascinating strategies to develop updated formulations, which improve efficacy, solubility, and bioavailability . HPβCD istituent . Previouositions . Accordi benefit .π–π, and ionic interaction, which prevents drugs from becoming crystallized [Recent progress in pharmaceutics has captivated scientists' attention toward improving drug solubility, stability, and dissolution using amino acids as coamorphous systems. The homogenous mixture forms intermolecular interaction, thereby increasing glass transition temperature (Tg), which enhances solubility and stability of the drug with the least excipient usage. Amino acid interacts with drugs via H-bond, tallized . Tryptoptallized  as it hoOverall, Tryp is comprehensively presumed to be nonpolar. As an essential amino acid, it contributes to the synthesis of key metabolic products such as protein, kynurenine, serotonin, tryptamine, melatonin, coenzymes, and niacin . The reaβCD and Tryp to improve its solubility and release patterns using a series of different w/w ratios via physical mixing (PM) and solvent evaporation (SE), followed by advanced experimental analyses. The bioactivity of complexes was deduced in animal models to validate our findings. Furthermore, the antioxidant activity of complexes was also investigated.To the best of our knowledge, there have been few studies on the ternary complexes of Glc for improving its solubility and release patterns. Hence, the present study is aimed at preparing the binary and ternary complexes of Glc with HPβ-cyclodextrin , DL-tryptophan , potassium dihydrogen phosphate , sodium hydroxide , magnesium stearate , Starch , polyvinylpyrrolidone K 25 , silica gel beads , and alloxan monohydrate . All the materials used in the present study were of analytical grade.The chemicals used in this study comprise Glc , methanol , hydroxypropyl-βCD) in binary systems in w/w ratios of 1 : 1, 1 : 2.5, 1 : 4, and 1 : 9 and three components (Glc: HPβCD : Tryp) in ternary systems in w/w ratios of 1 : 1 : 0.27, 1 : 1 : 1, 1 : 2.5 : 0.27, 1 : 3.6 : 3.6, 1 : 4 : 1, and 1 : 9 : 1, respectively. These ingredients were thoroughly ground, sieved (with a US 180 μm sieve), and stored in dried brown bottles. These samples were stored at 25°C in a desiccator [The physical mixtures were prepared using two components (Glc : HPsiccator .βCD was solubilized in deionized water (45% w/v), and Tryp dissolution was performed in aqueous methanol (70%). For the preparation of a 1 molar solution, 204 g of tryptophan was solubilized in 700 mL of methanol and 300 mL of distilled water. All the components were accurately and validly weighed in accordance with the previously stated ratios, and complexes were prepared using the conventional solvent evaporation method [Glc is insoluble in water and was dissolved in methanol (1 g/10 mL). HPn method .(1) Infrared Spectroscopic Studies. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra of pure Glc, HPβCD, Tryp, and all samples were obtained using an ATR-FTIR spectrometer  equipped with a mercury-cadmium-telluride detector. All samples were scanned at a resolution of 4 cm−1 over the wavenumber range of 400–4500 cm−1. Each spectrum was collected from 50 scans in the absorbance mode. Triplicate measurements were recorded, and mean values were used to ensure precision. In order to obtain FTIR spectra, the percent transmittance was plotted (y-axis) as a function of the wavenumber (x-axis). The spectral illustration was created using OriginPro 7.5 software.(2) Surface Morphological Study. The samples prepared by PM and SE methods with the ratios of 1 : 1, 1 : 4, 1 : 9, 1 : 1 : 1, 1 : 2.5 : 0.27, 1 : 3.6 : 3.6, and 1 : 9 : 1 were designated for surface morphology study. The surface morphology, shape, nature, and height of pure Glc, HPβCD, Tryp, and the selected complexes were investigated through a scanning electron microscope (Carl Zeiss Microscopy Ltd.). The sample was fixed on a brass stub before being coated with a thin layer of gold to ensure good electrical conductivity while performing microscopic scans. Images were captured at magnification powers of 200, 250, 500, 1000, 1500, and 2500 μm, with an accelerating voltage of 15 kV.(3) X-Ray Diffraction Studies. X-ray diffractograms of pure Glc, HPCD, Tryp, and samples  prepared by PM and SE were obtained using an X-ray diffractometer  with a HyPix-400 MF 2D hybrid pixel array detector at a voltage of 40 kV and a current of 30 mA. Cu was used as the targeting material. A modified system of diverging, receiving, and scattering slits of 1°, 0.2 mm, and 1° was utilized. The step angle was 0.05°, and the count time was 1 sec. The resulting patterns were recorded over a scan range of 5° to 50° at 2θ. OriginPro 7.5 software was used to create the comparison graphs.μm , and 1 mL of each solution was diluted to 10 mL with deionized water. It was diluted 3 times. The equilibrium solubility study was ultimately executed. The water solubility profile of binary and ternary complexes was analyzed through spectrophotometry at 226 nm [To determine solubility, 0.4 g of each sample containing binary and ternary complexes was added to 10 mL of deionized water. These mixtures were mixed thoroughly by a vortex mixer for 3–5 minutes and then kept on an orbital shaker at 150 rpm and 37°C for seven days to attain equilibrium. The samples were filtered through an AcrodiscGF filter syringe of 0.45 t 226 nm .(1) Micromeratic Properties. (a) Apparent Bulk Density. The precompressed powder was weighed, and volume was calculated using a volumetric cylinder. The following formula was used to calculate bulk density: bulk density = mass of powder/volume of powder.(b) Tapped Density. The precompressed and weighted powder was placed in a volumetric cylinder and tapped 100 times. Tapped density was calculated by the following formula: tapped density = mass of powder after tapping/volume of powder.(c) Compressibility Index. The compressibility index (CI) was calculated using the following formula: CI = tapped density–bulk density/tapped density.(d) Hausner's Ratio. Hausner's ratio was calculated by using the following formula: Hausner′s ratio = tapped density/bulk density(e) Angle of Repose. The angle of repose is determined by fixed funnel methods, in which the sample is allowed to accumulate and form a conical heap on the surface. The height and radius of the heap were calculated. Values are put below the equation, and the angle of repose was determined. tanθ = h/r.(1) Tablet Formation. A direct compression method was adopted for preparing tablets of Glc and its complexes. In all these tablets, Glc, starch, PVP (polyvinyl pyrrolidine), lactose, and magnesium stearate were added as binders, disintegrating agents, and lubricating agents. These were homogenously mixed and sieved through a mesh of US 180 μm. As shown in (2) Weight Variation, Hardness, and Friability. For performing the weight variation test, 20 tablets were weighed individually and the average weight for each sample was calculated. Each tablet's weight was compared to the average weight. The hardness of the tablets was tested using a Monsanto hardness tester. The tablet was subjected to strength by placing between anvils, and the crushing force was recorded. Friability is the percentage loss of tablets' weight due to mechanical action. It was determined using a Roche Friabilator. For this purpose, 20 tablets were selected for each sample. After finding their initial weight, these were put in Roche Friabilator. Tablets were revolved for 4 min at 25 rpm. After the completion of the cycles, the tablets were cleaned with a brush to remove any powder or crumbs [All values were expressed as mean ± standard error to the mean.in vitro drug release study on a pure drug, its binary and ternary complexes were undertaken using a paddle dissolution test apparatus. Each tablet sample was added to 900 mL of phosphate buffer solution (pH 7.4) as a dissolution medium at 37 ± 0.5°C. For making buffer, a 27.22 g of potassium dihydrogen phosphate solution was prepared in 1000 mL of deionized water. Similarly, a solution of 8 g sodium hydroxide was prepared in 1000 mL of deionized water. These solutions were mixed in 2000 mL of deionized water to make a 4 L buffer solution of pH 7.4. For carrying out the dissolution study of 20 tablets, 20 L buffer was prepared.In the The stirring speed was maintained at 100 rpm. In all the experiments, 10 mL of the sample was withdrawn from each vessel at a time interval of 5, 15, 30, 60, 90, 120, 180, and 240 minutes and replaced by adding an equal volume of fresh buffer at each time to maintain the sink condition. The concentration of drug released from each tablet sample was probed with an ultraviolet spectrophotometer using a quartz cell of 0.2 cm. The absorbance was taken at 226 nm [(1) Model-Dependent Approach. To know about the drug release phenomenon, different mathematical models such as the zero-order model, first-order model, Higuchi model, Hixson Crowell model, and Korsmeyer–Pappas model were used [ere used .(a) First-Order Kinetics. (b) Zero-Order Kinetics. (c) Hixson Crowell Model. (d) Higuchi Kinetic Model. (e) Korsmeyer–Peppas Model. t.” K = rate constant (kappa). Q0 = preliminary value of Q. n = release exponent.Q = amount of drug discharge in time “(2) Model-Independent Approach. The model-independent approach requires similarity (f2) and difference factor (f1). (1) Animals. Binary and ternary complexes of samples in different ratios  prepared by PM and SE methods were selected for the investigation of antidiabetic activity in animal models . The rabbits were maintained under standard conditions in a cage system and acclimatized for a period of 7 days prior to the commencement of the experiment. The rabbits were fed with a standard pellet diet and water ad libitum and kept at room temperature in a cycle of 12 hours of dark and 12 hours of light. All rabbits were categorized into 11 groups having six animals in each. All the animals used in this study were obtained from the university's animal house. The permission for the animal study was obtained from the biochemistry department's ethics committee, faculty of science, Bahauddin Zakariya University, Multan, Pakistan, under letter no. 545/PEC/2019. The experiment was carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986, and associated guidelines.(2) Diabetic Model. The fasting blood glucose level of each rabbit was monitored using an Accu-Chek glucometer. Then, diabetes was induced in the rabbits using alloxan monohydrate (150 mg/kg) dissolved in 10 mL of a 5% freshly prepared saline solution [solution . To makesolution . The rab(3) Administration of Samples. Diabetic rabbits were administered orally as solid pellets with10 mg/1.5 kg of pure Glc [βCD-Tryp to groups I to XI. The rabbits were grouped as follows: group I: control group treated with 0.01 g (10 mg) of Glc; group II: rabbits treated with 0.02 g of the sample (PM 1 : 1); group III: rabbits treated with 0.02 g of the sample (SE 1 : 1); group IV: rabbits treated with 0.1 g of the sample (PM 1 : 9); group V: rabbits treated with 0.1 g of the sample (SE 1 : 9); group VI: rabbits treated with 0.03 g of the sample (PM 1 : 1 : 1); group VII: rabbits treated with 0.03 g of the sample (SE 1 : 1 : 1); group VIII: rabbits treated with 0.08 g of the sample (PM 1 : 3.6 : 3.6); group IX: rabbits treated with 0.08 g of the sample (SE 1 : 3.6 : 3.6); group X: rabbits treated with 0.11 g of the sample (PM 1 : 9 : 1); and group XI: rabbits treated with 0.11 g of the sample (SE 1 : 9 : 1).pure Glc  with an Following administration, blood samples of the rabbits were collected from the marginal vein at 0, 1, 2, 3, 4, 6, 8, 12, and 24 hours. These blood samples were analyzed for glucose using the Accu-Chek glucometer. Mean and standard deviations were calculated. The following formula was used to calculate glucose reduction percentages:Antioxidant activity was determined for diabetic rabbits (group 0), group I, group V, and group XII. Blood samples were taken from the central ear artery and stored in heparin-containing syringes. From these blood samples, plasma and erythrocyte lysates (ELs) were prepared. After removing plasma, packed erythrocytes were washed twice with 9 g/L NaCl solution and hemolyzed with cold distilled water. In these hemolysates, EL-catalase and EL-SOD activities were determined immediately .μmol H2O2/min under specific conditions [EL-catalase activities were measured using a method developed by Beers and Sizer that is based upon the degradation of peroxide and was recorded spectrophotometrically at a wavelength of 240 nm. One unit of catalase is the amount of catalase that decomposes 1 nditions .EL-SOD activities were described following Beers and Sizer, in which the enzyme inhibits autooxidation of epinephrine and was recorded spectrophotometrically at 480 nm. One unit of SOD is defined as the amount of SOD that inhibits 50% of the autooxidation of epinephrine .The plasma total antioxidant status (TAS) (mmol Trolox eq/L) was measured using the Erel spectrophotometric method , which iU test, followed by Student's t-test and Kruskal–Wallis test. Using Spearman's correlation test, correlation analysis was achieved.All the values of the current data are expressed as mean ± SD. The experiment results were assessed using the ANOVA  single factor by SPSS-16. Furthermore, in order to compare which group of samples differs significantly, Tukey's multiple comparison test (for solubility and drug release test) was applied. Group comparisons in antioxidant activity were determined by the Mann–Whitney −1 corresponding to the –NH stretch of urea moiety, 1162.11 cm−1 due to symmetric stretching of S=O, and 1349.68 cm−1 due to asymmetric stretching of S=O. C=O stretching of carbonyl was observed between 1700 cm−1 and 1709 cm−1. –CH stretching peaks due to the aromatic ring of Glc were detected at 2935.87 cm−1 [βCD indicated the –OH group in the wavenumber region of 3200 cm−1 to 3600 cm−1. C=O stretching vibrations were detected at 1031.63 cm−1. Our findings adequately showed the –CH peak at 2929.75 cm−1 [−1 due to –CO2 asymmetric stretch, 1453.66 cm−1 due to –CH mode of the indole ring, and 1582.10 cm−1 due to amide bond. Data showed that –NH stretching vibration was observed at 3400.71 cm−1 [Characteristic bands of pure Glc were observed at 3276.34 cm.87 cm−1 . These wβCD and Tryp made by PM and SE are eminently presented in −1and a significant reduction in intensity in all SE complexes as compared to PM complexes and pure Glc. The peaks due to the –NH group of Glc and –OH group of HPβCD at 3200 cm−1–3600 cm−1 were lost in the complexes of Glc and HPβCD in binary and ternary systems made by PM and SE. These showed bonding interactions among the –NH group of Glc and the –OH group of HPβCD, with altered frequencies of S=O, CO, and –CH stretching peaks of Glc. It should have appeared as a sharp shoulder in the spectrum of complexes if it was not altered by the complexation process. A completely different pattern of absorption bands was observed in the fingerprint region, including shifts of some bands, such as 1349.68 cm−1. The obtained IR spectra for Glc and HPβCD in binary and ternary complexes indicated slight modifications in comparison to those of free Glc and were generally consistent with previous reports on Glc and HPβCD complexes [βCD complexes, it could be concluded that the Glc-HPβCD complex was formed.ATR-FTIR spectra of Glc and its binary and ternary complexes with HPomplexes , 36. Sin−1 (−CH mode of indole ring) was observed to be decreased after complex formation in ternary complexes. Peak intensities due to the –NH stretch, –SO stretch, and –CH stretch of Glc and the CO of HPβCD were also found to be altered. The band at 1658 cm−1 representing the NH3+ asymmetric angular deformation disappeared in the inclusion complexes, and an intense band appeared at 1632 cm−1. This was related to the new chemical environment of amino acids, in which NH3+ formed a hydrogen bond with –OH groups of HPβCD. Our results are consistent with those of a previous study [−1 significantly decreased after complexation as compared to the physical mixture, with SE 1 : 9 : 1 yielding the best results. This decrease in respective intensities at 1582 cm−1 and 1453 cm−1 proved that tryptophan had successfully formed inclusion complexes with Glc and HPβCD. Thus, as evidenced by our findings, these changes support the complex formations between the drug and carriers.The intensity of the band at 1453.66 cmus study . Similarθ having intensity at 10.4° (2168), 14.9° (922), 17° (1245), 18° (1706), 20.7° (1024), 22° (762), and 26.2° (402). The highest peak was formed at 10.4°, which had an intensity of 2168. The Glc showed high-intensity peaks due to its crystalline nature  is shown . Crystalβ-CD polymer. Our results are comparable to Glc and β cyclodextrin complexes [βCD in complex formation. An increase in intensity was observed more in 1 : 1 : 1 than in 1 : 9, which further validates the rise in the crystallinity nature. The least intensity was observed in 1 : 9 : 1. Thus, peak intensity decreases as the carrier's ratio increases. At 22° intensity, the peak in complexes was found to be totally diminished, which proved the interaction between the drug and both carriers.The 1 : 4 binary complexes made by physical mixture revealed the same intensity changes as SE 1 : 1. A decrease in intensity of some peaks with an increasing carrier ratio in binary complexes authenticated the amorphousness of the complexes. This showed an interaction between Glc and HP-omplexes . CrystalFurthermore, the complexes with notably lower intensities had a lower number of signals, implying that the complexes were more amorphous than the drug. The extent of crystallinity influences the dissolution of the drug. An amorphous or metastable form will dissolve at the fastest rate because of its higher internal energy and greater molecular motion, which enhances the thermodynamic properties compared to crystalline materials .Some changes in the peak position of Glc were adequately observed in PM and SE binary and ternary complexes. Pure Glc has prominent peaks at 10.4°, 14.9°, 17°, 18°, 20.7°, 22°, etc. They were clearly spotted at the same position in PM and SE, but the intensities were observed to be significantly decreased. In complexes made by SE, the intensity of Glc peaks was significantly reduced. We can deduce from the previous observations that the drug's crystalline nature was preserved. However, the relative reduction of the diffraction intensity of Glc in complexes indicates that the quality of the crystals was reduced. Some new diffraction peaks were also observed in complexes made by SE, which proves the formation of inclusion complexes. The appearance of new diffraction peaks in the spectrum of the “interacted mixture,” as well as the shift of the guest molecules' characteristic peaks and changes in their relative intensity, all indicate the formation of a new solid phase and support the actual inclusion complex formation . The resμm, ×250 100 μm, ×500 50 μm, ×1000 10 μm, ×1500 10 μm, and ×2500 10 μm. Glc was found to possess irregularly shaped crystals of different sizes. Lamellar-shaped crystals were also observed at this resolution. Small crystals were found to stick to large, irregular, and lamellar-shaped crystals  made by PM and SE showed random crystals of lamellar, globular, thin, and flaky structures. With the increasing ratio of carriers in complexes, the crystalline nature of components was presumed to be reduced. Agglomeration was clearly visible at high resolution. Thus, it evidenced the complexation of pure Glc with both carriers. SEM images of PM and SE of 1 : 9 : 1 demonstrated much-reduced crystallinity than 1 : 3.6 : 3.6 and other binary complexes. Increased carrier ratios in complexes lead to categorically reduced crystallinity  can be made more soluble by forming inclusion complexes with HPbutylβCD .p < 0.05). Likewise, ternary complexes made by the SE method demonstrated higher solubility (p < 0.001) than binary complexes (p < 0.05) as compared to pure Glc. The concentration of the drug released from binary complexes of physical mixtures was observed between 7.31 ± 0.50 to 10.7 ± 0.60 mg/mL, and that of SE was between 8.36 ± 1.20 and 12.69 ± 0.60 mg/mL.The Glc solubility was equitably recorded in 0.5 mg/mL of deionized water at 37°C. According to graphical representation , complexβCD and βCD increased the solubility of difenoconazole in water [The concentration of drug released from ternary complexes made by physical mixtures was discerned at 11.75 ± 0.90–17.4 ± 1.00 mg/mL, while that of SE was found to be 13.2 ± 0.90 to 18.73 ± 1.00 mg/mL. Ternary complexes were more soluble (16.9–18.23 times in 1 : 9 : 1) than binary complexes (10.2–12.9 times in 1 : 9). The solubility was determined in the descending order of SE > PM > pure Glc, which was substantially all inclusive. Of all the preparations, 1 : 9 : 1 showed the best solubility profile. When compared to pure Glc, the concentration of drugs released from PM and SE 1 : 9 : 1 was 17.40 ± 1.0 and 18.73 ± 1.0, respectively. Recent studies found that HPin water , 46. In in water .PM and SE mixtures of all samples showed a substantial increase in equilibrium solubility as the carrier ratio increased. The increase in solubility was due to the amorphous nature of the prepared samples or the inhibition of crystallization. Generally, the solubility profile of binary and ternary complexes prepared by PM and SE methods agreed with the data of XRD, FTIR, and SEM, which indicated that both PM and SE improved the solubility profile of Glc.(1) Micromeritics of the Powder Sample. The powder mixture of all batches showed good flow characteristics (eristics . Bulk de(1) Weight Variation, Hardness, and Percentage Friability of Tablets. For each sample, 20 tablets were prepared and evaluated assiduously for weight variation, hardness, and friability. For this purpose, their average and standard deviation were determined. All the tablets had a weight variation of 199.2 ± 1.06 mg to 199.74 ± 0.90 mg. The hardness of tablets was observed at 2.8 ± 0.68 to 4.27 ± 0.81 kg/cm2. The tablets showed less than 1% friability, which manifests good mechanical characteristics .The binary and ternary complexes of Glc with HPβCD and Tryp exhibited considerably enhanced drug release compared to pure Glc and PM. From all samples, 1 : 9 : 1 showed a maximum drug release percentage. When compared to pure Glc, it showed 65.4 ± 1.0 and 77.40 ± 1.0 mean percent drug release. All preparations showed a substantial increase in the percent drug release (Binary and ternary complexes formed by SE demonstrated maximum drug release (36 ± 1.0%–77.4 ± 0.05%) compared to PM. Thus, ternary complexes made by the SE method demonstrated a significantly higher drug release profile than PM mixtures when compared to pure Glc. A graphical comparison between pure Glc and binary and ternary systems in different polymer ratios is shown . The SE  release , which cβCD in the form of inclusion complexes with difenoconazole and thiophanate methyl demonstrated a faster dissolution than pure drugs [R2), it was determined that the majority of the formulations follows the Higuchi and Korsmeyer-Peppas models. All findings were evaluated using ANOVA (one way), which showed that there is a significant difference between formulations (p ≤ 0.043).Research studies have reported that ternary complexes of the model drug naproxen-proline with either arginine or tryptophan have increased solubility and dissolution profile . In compre drugs , 49. Thest hour following Alloxan, the values did not vary significantly from the fasting values. The values at the 2nd and 3rd hours were significantly higher (p ≤ 0.05), followed by a decline at the 4th hour (5.4 ± 0.11 mmol/L to 7.8 ± 0.16 mmol/L) and 6th hour (5.24 ± 0.17 mmol/L to 8.7 ± 0.11 mmol/L) when compared to the 2nd hour (5.97 ± 0.12 mmol/L to 7.62 ± 0.17 mmol/L), but were then significantly higher at the 8th hour (5.23 ± 0.11 mmol/L to 9.35 ± 0.33 mg/dL). All these values were significantly higher (p ≤ 0.05) when compared to the initial fasting blood glucose level. From the 8th hour posttreatment, the blood glucose level increased significantly up to 48 hours (p ≤ 0.05). Thus, animal models with blood glucose levels > 11.11 mmol/L were presumed to be diabetic  and in the form of complexes. Glc (group I) treatment alone significantly increases the level of TAS, EL-CAT, and EL-SOD. However, when combined with HPβCD in a binary system (group V), these antioxidant markers increased compared to those of group I. HPβCD acts as a secondary antioxidant. It augmented the antioxidant activity of Glc in diabetic rabbits. Group XII (ternary complex) achieved the best results with a p value less than 0.001  revealed a marked decrease in plasma TAS, EL-CAT, and EL-SOD levels, which were restored by Glc alone (an 0.001  because in vitro [The increased formation of reactive oxygen species (ROS) occurs under diabetes conditions, affecting antioxidant status. This oxidative damage may play a vital role in the development of major macro- and microvascular diseases like atherosclerosis. Lipid peroxide levels increase in diabetic patients' circulatory systems. Acute glucose levels have been linked to lipid peroxidation, and recent investigations have shown that this glucose load reduces the human antioxidant defense mechanism. Glc inhibits LDL oxidation in vitro . The marin vitro , who obsβCD acts as a secondary antioxidant, increasing the system's natural antioxidant capacity. Furthermore, this study found that substrates have antioxidant properties embedded in the hydrophobic moiety of cyclodextrin, resulting in the nonavailability of these natural antioxidants to the oxidative enzymes [A previous study showed that HP enzymes .Hoseini et al. investigated the effects of dietary Tryp on the oxidative condition of the fish's intestinal mucosa. They concluded that consuming this amino acid at the recommended dietary amount could improve the antioxidant status in animal models .βCD, and Tryp possess antioxidant activity. The combination of Glc therapy with both carriers in the form of complexes contributes to the control of oxidative stress and demonstrated better outcomes in improving antioxidant parameters in diabetic patients. This combination of the drug with both carriers may improve diabetic patients' survival rates and has implications for further clinical trials.Glc, HPβ-CD, and Tryp complexes, as well as to compare the efficiency of binary and ternary complexes. Furthermore, our research can be viewed as a type of screening in which we selected the best ratio and method for achieving the par excellence results. The binary and ternary complexes of Glc with HPβCD and Tryp were prepared using the PM and SE methods. The possibility to use the SE method enables more accurate product definitions.To conclude, the value of this work has been to discuss the methods of preparation, solubility, drug release profile, bioactivity, and antioxidant activity of Glc, HP-in vitro drug release profile can be improved as the carrier ratio increases in complexes. The FTIR results showed that adequate complex formation occurred between Glc and carriers. According to X-ray diffraction and SEM image studies, increasing the carrier ratio causes the crystalline nature of Glc to change to amorphousness, substantiating the complexation phenomenon between pure Glc and carriers. Additionally, an antioxidant study revealed that Glc, along with both carriers, has a high potential as a free radical scavenger and may improve TAS, SOD, and CAT levels, which are reduced in diabetics. This could substantiate complexes' secondary benefit. The in vivo antidiabetic activity of pure Glc and the complexes indicates that Glc's binary and ternary systems may improve antihyperglycemic activity while shortening its duration of action. Our findings indicate that increasing the ratios of Tryp and HPβCD in a constant amount of drug may restore and improve Glc's carrier-binding and medicinal properties. Our findings showed that PM and SE 1 : 9 : 1 performed well in all preparations when compared to the other binary and ternary complexes. Thus, cyclodextrins and amino acids have the potential to serve as the foundation for a new platform technique to overcome the challenges associated with poorly soluble drugs. Our findings warrant future studies of Glc solubility with other amino acids, as well as additional animal studies and clinical trials.Ternary complexes prepared using the SE method showed a higher solubility than pure Glc and other binary complexes. The


Alternaria solani and is an increasing problem worldwide. The primary strategy to control the disease is applying fungicides such as succinate dehydrogenase inhibitors (SDHI). SDHI‐resistant strains, showing reduced sensitivity to treatments, appeared in Germany in 2013, shortly after the introduction of SDHIs. Two primary mutations in the SDH complex (SdhB‐H278Y and SdhC‐H134R) have been frequently found throughout Europe. How these resistances arose and spread, and whether they are linked to other genomic features, remains unknown. For this project, we performed whole‐genome sequencing for 48 A. solani isolates from potato fields across Europe to better characterize the pathogen's genetic diversity in general and understand the development and spread of the genetic mutations that lead to SDHI resistance. The isolates can be grouped into seven genotypes. These genotypes do not show a geographical pattern but appear spread throughout Europe. We found clear evidence for recombination on the genome, and the observed admixtures might indicate a higher adaptive potential of the fungus than previously thought. Yet, we cannot link the observed recombination events to different Sdh mutations. The same Sdh mutations appear in different, non‐admixed genetic backgrounds; therefore, we conclude they arose independently. Our research gives insights into the genetic diversity of A. solani on a genome level. The mixed occurrence of different genotypes, apparent admixture in the populations, and evidence for recombination indicate higher genomic complexity than anticipated. The conclusion that SDHI tolerance arose multiple times independently has important implications for future fungicide resistance management strategies. These should not solely focus on preventing the spread of isolates between locations but also on limiting population size and the selective pressure posed by fungicides in a given field to avoid the rise of new mutations in other genetic backgrounds.Early blight of potato is caused by the fungal pathogen  If the weather conditions are favourable and A. solani remains uncontrolled, yield losses can reach up to 40% , after wheat, maize, and barley (Eurostat 2020). To guarantee the yield, farmers adhere to good agricultural practices, for example, crop rotation and sufficient nutrient supply, but in most cases the application of fungicides is needed.Early blight is one of the major diseases in potato‐growing areas. The causal agent of early blight is the fungal pathogen % Kapsa, , 2014. T% Kapsa, . Due to % Kapsa,  predicteFor early blight control, three main fungicide groups are available: the Quinone outside Inhibitors (QoIs), the Succinate Dehydrogenase Inhibitors (SDHIs), and the Demethylation Inhibitors (DMIs), of which QoIs and SDHIs are most widely used and until recently considered most effective.A. solani and other fungi for all of these fungicide groups. DMI resistance appears to be related to changes in expression of the target site, Cyp51, possibly linked with a mutation  primers . A single spore was collected from each isolate and propagated under sterile condition for further usage.Forty‐eight different isolates were collected from five different localities across Europe Figure . All sam2.2A. solani cultures, grown on SNA. Liquid cultures were incubated for 48 hours at 28°C on a rotary shaker (110 rpm). Afterwards, mycelium was filtered through a cheese cloth and squeezed to remove most of the liquid. After freeze drying, mycelium was grinded with liquid nitrogen and a bit of clean sea sand to break the cells most efficiently. 50–100 mg of lyophilized powder was added to 1 ml of DNA extraction buffer , nucleic lysis buffer , and sarkosyl (10%) in a ratio of 2.5:2.5:1), 20µl proteinase K (25 mg/ml), and 20µl RNAse A (20 mg/ml) in a 2 ml tube. Everything was mixed well and incubated for 1hour at 60°C – inverting the tubes occasionally to gently mix the content. Afterwards samples were centrifuged for 10 minutes at maximum speed, and the supernatant was transferred into a new tube. An equal amount of PCI ) was added to the tube and incubated overnight on a rotary shaker at 4°C (gentle rotation to avoid damaging DNA). On the following day, samples were spun down for 15 minutes at maximum speed and upper phase was transferred to a new tube. An equal amount of SEVAG ) was added and tubes were incubated for at least 1 hour on a rotary shaker at 4°C (gentle rotation). In a next step, samples were centrifuged for 10 minutes at maximum speed and supernatant was transferred to a new tube. For precipitation, 0.7 volume of isopropanol (room temperature) was added and samples were spun down for 15 minutes at 9,600 g. After washing the DNA pellet with 70% ethanol, remaining liquid was removed by pipetting. To entirely remove the ethanol, tubes were placed in a heating block at 37°C to let them dry completely. DNA pellet was dissolved in 100 µl distilled water and stored at 4°C. A Qubit was used to determine the quantity of the extracted DNA. Sequencing libraries were prepared by BGI Ltd (Hong Kong) using their proprietary protocol. The sequencing was done on the DNBSEQ platform, using paired end 150 bp reads, aiming for up to 3 Gb of data per sample.For high‐quality DNA extraction, 200 ml of potato dextrose broth was inoculated with small agar plugs from 10 to 14 days old 2.3The quality of the raw sequence data was checked using FastQC version 0.11.5 Andrews, 2.4The vcfR package (version 1.12.0) was used to read and visualize the vcf file for quality control , rbp2 (KC584430), and tef1 (KC584688) were extracted form NCBI search against the reference genome using BLAST+ , RBP2 , TEF1  for A. dauci and A. porri respectively). To analyse the genomic regions coding for the SDH subunits, we performed a BLAST search of sequences each of the SDH subunits against the A. solani reference and used the identified start and stop site to extract alternative reference fasta files using GATK  . To achieve that, vcf chunks of chromosomes were imported separately and analysed discretely. Therefore, a function was defined to allow the analysis of each chromosome and to re‐aggregate generated data, subsequently. This is necessary since the native function for data‐import (readVCF) does not apply for haploid datasets. Segregating sites per window were extracted and normalized per site. ggplot2 was used to visualize our findings.2.7We extracted the SNPs as the alternative reference genomes using the GATK FastaAlternateReferenceMaker tool to create an alignment of the whole isolates’ genomes and to construct a population phylogeny  was performed using the R packages gdsfmt (version 2.18.0) and SNPRelate (version 1.18.1)  was performed and a cross‐entropy criterion was calculated to identify the best statistical model describing ancestral populations of the dataset. The minimal cross‐entropy for the dataset was determined with multiple repetitions . Admixture analysis was performed for k=6, k=7, and k=8 [Q))] and ordered according to individually assigned genotypes or sample origin. Visualization and inference with mutations was done using the tidyverse package (version 1.3.1)   analysis was performed using poppR (version 2)  .An SNP distance matrix was constructed from the alternative reference sequences with the extract alternative reference fasta option from GATK . SNP call files are deposited to Zenodo.org under doi 10.5281/ , 7 from Southern Germany (Bavaria), 8 from Serbia, and 12 from Sweden  confirmed that BE_SL002 is A. solani. With this combination of barcodes, we can define two haplotypes  SNPs between all isolates. Due to the comparably large number of unique SNPs in BE_SL002, further analyses were done without this sample, to allow better comparison.We found a transition versus transversion (ts/tv) ratio of 3.05. Forty‐eight percent of the SNPs are singletons. The Site Frequency Spectrum (SFS) for the sample set shows a homogenous decrease first. However, the overall number of SNPs is not decreasing continuously, in fact, some minor peaks are occurring for higher SNP frequencies Figure . This inSdhB), 2 (SdhC), and 5 (SdhD). Thus, the fungicide target genes are not located specifically on very conserved or very diverse chromosomes.On average we found one SNP every ~241 bp. SNP density varies per chromosome, ranging from one every 197 bp for chromosome 4 to one every 277 bp for chromosome 2. Using sliding window analysis with 10kb windows, confirmed that SNP‐density varies along the chromosomes and is highest and lowest in chromosome 6 and 1 respectively Figure . The dif3.2Next, we wanted to know whether the genetic variation is similar for each of the five sample locations in Europe, and in our US samples. To this end, we summarized all statistics per location Table . Even exIn line with the higher number of SNPs, we also found that the Belgian samples have the highest nucleotide diversity (Watterson's θ or π). Interestingly, we found some variation in Tajima's D. However, Tajima's D is negative or close to 0 in most localities. This indicates a relatively high number of singletons and is in line with the overall observations from the SFS. However, a Tajima's D of 1.024 in Sweden seems to violate the assumption of neutral theory suggesting less low‐frequency polymorphisms, probably due to higher clonality in these samples.3.3A. solani is considered to be an asexual pathogen, but some reports suggest parasexual reproduction is occurring in different populations and maybe even between related species . To estimate the most likely number of ancestral populations of the dataset, the cross‐entropy criterion was calculated. The lowest cross‐entropy was observed at K=7 Figure , thus add Figure , Table 1Next, we assigned each isolate to the respectively most dominant of the seven genotype groups and calculated diversity statistics for each of them Table . The nucSeeing the limited diversity in some genotype groups, we asked how many isolates could be considered clones of each other. To this extent we created a pairwise SNP‐distance table for isolates Table . This reA. solani genotypes would show regional clustering. Variation in the diversity within geographical regions can also be observed. The samples from Belgium and Sweden show the most complex admixture structure, followed by Bavaria. The high complexity in Sweden is particularly interesting, because in absolute number of segregating sites, this population did not appear to be the most diverse. Interestingly, some of the clones described above can be found on different sides of the European continent  or even across continents (US_JW002 clusters with Serbian and Swedish isolates).To better visualize the diversity per location, we also sorted the isolates per locality Figure . It shou3.5To understand the rise and spread of SHDI fungicide resistance, we overlaid the genotyping data with our data on SDHI‐target mutations. The H134R and H278Y mutations occur 15 and 10 times in our dataset, respectively. H134R is found in every locality, except Serbia; H278Y can be found in all locations but Serbia and the United States. Furthermore, we found three mutations  that occur only once in the dataset. In 20 samples, we found no mutation related to SDHI fungicides. Mutation‐free isolates were collected in all different localities except for the United States.sdh SNPs were found in different genetic backgrounds  based on all SNP data Figure . These nA. solani , we expected to see certain geographical patterns related to the genetic diversity, for example, patterns of isolation by distance or predominance of a certain genotype in a locality. The strongest geographical patterns can be observed in invasive pathogens, like the rice and wheat blast fungus Magnaporthe oryzae, where multiple genetic lineages can be found in Southeast Asia, but single invasive lineages spread elsewhere and sometimes became epidemic ‐based study with populations from China. Assumed clones were detected in multiple populations separated by thousands of kilometres and random association among loci was found in half of the populations assayed , and then spread. However, recent work showed that the F129L mutation in the cytochrome b target gene responsible for this resistance could since 2016 also be observed in another genotype sexual recombination needs to happen in the field in order to explain the observed diversity patterns (Alvarenga et al., A. solani isolates under certain conditions or on a certain host cultivar alone might not be the only determinants for A. solani diversity in the field. Early blight lesions often appear to contain a mix of Alternaria species. A. solani often co‐occurs with A. alternata (e.g., Zheng & Wu, A. solani, A. linariae, and A. grandis, and the small spored A. alternata (Bessadat et al., of A. alternata (Ding et al., A. alternata possessed a greater diversity in the field (Ding et al., A. solani isolates were more severe and A. solani is the more dominant partner in the interaction. Studies on Alternaria leaf spot on rape seed showed that also this disease is not caused just by A. brassicae, but that it can also be caused by A. japonica and that the ratio between the species and aggressiveness of the species was temperature dependent (Al‐lami et al., A. solani can potentially also be shaped by the presence of diverse Alternaria species and that further research is needed to understand the effects of coinfection on early blight epidemiology. The presence of different co‐infecting Alternaria spp. might cause different evolutionary pressure on the isolates.Fungicide pressure and the aggressiveness of A. solani in Europe. We revealed surprising genetic diversity patterns throughout Europe and show that fungicide resistance evolved multiple times independently, rather than evolving once followed by spreading. These findings can help inform policy and fungicide management practices. The finding of multiple fungicide resistance mutation events highlights that there is “not one person to blame” for the rise of fungicide resistance in A. solani. This emphasizes the need to reduce the evolutionary pressure in each individual field and keeping the population size as small as possible at all times (McDonald & Linde, A. solani epidemics. To do this without increasing too much the evolutionary pressure in the field, fungicide application time frames and quantities need to be carefully adjusted as they play an important role in the development of resistances and diseases. Unfortunately, in our dataset, loss of fungicide sensitivity cannot be linked to the degree of fungicide‐use, since data is not fully available for all localities in this project.We presented a first genome‐wide diversity analysis for the early blight pathogen A. solani dispersal patterns over long distances will become increasingly more relevant. Recent studies have shown clear differences in aggressiveness in different A. solani isolates in general, for example, not associated with specific fungicide target mutations (Mphahlele et al., A. solani over time, which will lead to a better understanding of pathogen dispersal patterns and should contribute to improving management strategies.Our analysis also shows that some true clonal isolates can be found thousands of kilometres apart. Understanding The authors have no conflict of interest.Fig S1‐S7Click here for additional data file.Table S1Click here for additional data file.


Marine mammals are under pressure from multiple threats, such as global climate change, bycatch, and vessel collisions. In this context, more frequent and spatially extensive surveys for abundance and distribution studies are necessary to inform conservation efforts. Marine mammal surveys have been performed visually from land, ships, and aircraft. These methods can be costly, logistically challenging in remote locations, dangerous to researchers, and disturbing to the animals. The growing use of imagery from satellite and unoccupied aerial systems (UAS) can help address some of these challenges, complementing crewed surveys and allowing for more frequent and evenly distributed surveys, especially for remote locations. However, manual counts in satellite and UAS imagery remain time and labor intensive, but the automation of image analyses offers promising solutions. Here, we reviewed the literature for automated methods applied to detect marine mammals in satellite and UAS imagery. The performance of studies is quantitatively compared with metrics that evaluate false positives and false negatives from automated detection against manual counts of animals, which allows for a better assessment of the impact of miscounts in conservation contexts. In general, methods that relied solely on statistical differences in the spectral responses of animals and their surroundings performed worse than studies that used convolutional neural networks (CNN). Despite mixed results, CNN showed promise, and its use and evaluation should continue. Overall, while automation can reduce time and labor, more research is needed to improve the accuracy of automated counts. With the current state of knowledge, it is best to use semi-automated approaches that involve user revision of the output. These approaches currently enable the best tradeoff between time effort and detection accuracy. Based on our analysis, we identified thermal infrared UAS imagery as a future research avenue for marine mammal detection and also recommend the further exploration of object-based image analysis (OBIA). Our analysis also showed that past studies have focused on the automated detection of baleen whales and pinnipeds and that there is a gap in studies looking at toothed whales, polar bears, sirenians, and mustelids. In thise.g.,i.e., sub-meter spatial resolution) allows monitoring marine mammals in remote areas  rookery. Furthermore, UAS collect multispectral imagery of higher resolution than satellites, at centimeter . Moreover, UAS can provide thermal infrared (IR) imagery at sufficient resolution to detect marine mammals on land  imagery have surged as complementary tools to count individuals of various taxa of marine mammals  took 24 h to be analyzed with three replicates (eight hours per person) for the work of 2. Furthermore, 2 at a 1:1,500 m. scale (2 min per km2), and 2 . While satellite images have allowed continental-scale abundance and distribution studies in remote locations  classify individual pixels into animal or background classes based on spectral information or texture, object-based methods classify groups of homogeneous pixels into the different classes, which allows for the use of other variables, such as size and shape  Primo database was used repeatedly between August 2019 to January 2022 to identify peer-reviewed literature, conference proceedings, grey literature, and theses and dissertations about combinations of keywords such as “marine mammals”, “remote sensing”, “UAV”, “UAS”, “satellite” and “images”. The Primo database searches across the majority of the authoritative electronic and print resources subscribed to and purchased by the University of Florida Libraries  (Ursus maritimus)  than only spectral responses, such as deep learning or object-based image analysis (OBIA).We found 13 studies on automated detection of marine mammals ranging from 2013 to 2021, of which five used UAS imagery and eight used satellite imagery . While mstralis)  and polaritimus) . Histogrritimus) . Automatritimus) . These mritimus) . This alLobodon carcinophaga), Weddell seals (Leptonychotes weddellii), leopard seals (Hydrurga leptonyx) and Ross seals (Omnatophoca rossii) study. Arctocephalus pusillus) detection. Convolutional neural networks (CNN), a deep learning approach, have been increasingly used to analyze satellite and UAS images of whale hotspots  and pinnArctocephalus pusillus), but also loggerhead sea turtles (Caretta caretta), and Australian gannets (Morus serrator) in UAS imagery from separate sites. The authors reference the aggregation of pups in creches as one of the factors that made seal detection difficult compared to that of the other taxa analyzed, up to the point that the CNN was not able to distinguish between multiple aggregated pups. Whales can also offer particular detection challenges based on their behavior. For instance, i.e., with their peduncle emerged and breaching, were part of most of the true positive cases along with blowing and logging. However, they recognized that the spyhopping behavior compromised the CNN’s ability to detect whales.CNN studies have also sparked a discussion on how marine mammal behavior impacts detection abilities. For example, i.e., only those visible in the imagery. This is a common limitation of visual surveys from land, ships, aircraft, or using UAS or satellite imagery, and especially in marine mammal taxa that are permanently in the water . While, as mentioned before, UAS and satellite imagery have the advantage of allowing surveyors to go over them many times and cross-validate their counts with other researchers—helping to minimize perception bias compared to field surveys from land, ships or aircraft—they are still visual methods not able to detect animals submerged to the point of not being visible. Consequently, it is worth noting that while this review is focused on the automated detection of marine mammals in imagery to obtain animal counts as a first step, further steps are needed after the counts to calculate abundance or density estimates. For example, there is a requirement to make adjustements for submerged animals not showing in the imagery—animals available in the study area but not at the surface (not detectable), as e.g., In terms of detectability and accuracy evaluation, while The general approach presented above—comparing objects detected automatically with user observations assumed to correspond to all animals detectable in the image—aligns with evaluation methods commonly used in remote sensing. When adopting this approach, false negatives correspond to detectable animals missed by the computer. In contrast, false positives refer to anything that is not the detection target, such as water or rocks, and is classified wrongly as a marine mammal by the computer. False negatives thus contribute to an undercount of marine mammals, and false positives contribute to an overcount. However, there is currently a lack of consistency in accuracy metrics to evaluate automated methods across animal detection studies . FurtherEquation 1: F1 index as defined by Equation 2: F1 index as defined by e.g., texture)  , they artexture) . OBIA fitexture) . This prtexture) . In theoi.e., i.e.,Eubalaena australis), Odobenus rosmarus), but this was dependent on site heterogeneity .Some studies have used object features, such as shape i.e., , and a sOBIA has not been used extensively to detect other animals either, leaving pixel-based studies as predominant. While OBIA has been suggested to perform better than pixel-based analyses in land classification, that is still not clear in the examples of OBIA for counting animals see . It is ai.e., i.e., cetaceans and sireneans) remains a knowledge gap at this time.Another resource worth exploring in the future for marine mammal automated image analysis is the use of thermal IR. The combined use of shape, size, and temperature captured from thermal IR allowed Here we assess the number of false negatives in the different studies of False negative rate (Equation 3): ratio between the number of animals missed by an automated method (numerator) and the number of animals detectable in the image  (denominator), presented as a percentage. FN: false negatives. TP: true positives. False positive over detectable rate : ratio bTo summarize these two metrics into one, in the same way the F1 index integrates precision and recall, we suggest the automated count deviation . The aute.g., e.g.,Furthermore, we note that even though manual counts are taken as a reference of the animals detectable in the image, this is not an infallible strategy. The effects of environmental conditions, UAS flight-related variables, and the angle of imagery capture on manual detection certainty of different species should continue to be investigated see . FurtherAutomated count deviation : ratio be.g., Enhydra lutris) or dolphins of the Cephalorhynchus genus. Nevertheless, the manual analysis of satellite and UAS imagery is time- and effort-intensive, for which automation can help , as well as the relative scarcity of archival satellite imagery over sea compared to terrestrial habitats, leaving researchers only with more expensive tasking options. Automated methods have the potential to complement airplane and vessel marine mammal surveys, helping overcome old limitations, such as higher coverage in remote regions. However, to do that, satellite imagery needs to become more available and affordable further away from the coast, and high-endurance UAS need to become more affordable too, while their regulation needs to be flexibilized. In this context, joint initiatives for further research are necessary to not only to exchange imagery, but also to develop automated methods that both save researchers time and provide accurate counts, while being versatile enough to work in different settings and distinguish coexistent species.Marine mammal abundance and distribution studies can be well served from a combination of different surveying methods. For instance, UAS and satellite images, airplane and vessel surveys, to detect animals in the surface; passive acoustic monitoring, to detect submerged animals’ vocalizations e.g., ; and sat10.7717/peerj.13540/supp-1Table S1Number of true positives (TP), false positives (FP) and false negatives (FN) used in our calculations for Equations 3, 4 and 5 in Table 1, together with the source in each study from which they were extracted directly or calculated from.Click here for additional data file.


The pandemic of coronavirus disease 2019 (COVID-19) led to a series of strict control measures in Chinese cities, resulting in a short-term significant improvement in air quality. This is a perfect case to explore driving factors affecting the PM2.5 distributions in Chinese cities, thus helping form better policies for future PM2.5 mitigation. Based on panel data of 332 cities, we analyzed the function of natural and anthropogenic factors to PM2.5 pollution by applying the geographically and temporally weighted regression (GTWR) model. We found that the PM2.5 concentration of 84.3% of cities decreased after lockdown. Spatially, in the winter of 2020, cities with high PM2.5 concentrations were mainly distributed in Northeast China, the North China Plain and the Tarim Basin. Higher temperature, wind speed and relative humidity were easier to promote haze pollution in northwest of the country, where enhanced surface pressure decreased PM2.5 concentrations. Furthermore, the intensity of trip activities (ITAs) had a significant positive effect on PM2.5 pollution in Northwest and Central China. The number of daily pollutant operating vents of key polluting enterprises in the industrial sector (VOI) in northern cities was positively correlated with the PM2.5 concentration; inversely, the number of daily pollutant operating vents of key polluting enterprises in the power sector (VOP) imposed a negative effect on the PM2.5 concentration in these regions. This work provides some implications for regional air quality improvement policies of Chinese cities in wintertime.Fine particulate matter (PM These measures brought a temporary halt on human activities (2.5) decreased by 10.5%  model was adopted to quantitatively evaluate the spatiotemporal heterogeneous in the relevance between driving factors and PM2.5 in the short term. Therefore, we could distinguish the main driving factors that dominate the temporal and spatial distribution of PM2.5 concentration in different cities in winter, the season with the most severe haze. That could provide a reference for the local government to put forward more effective haze control policies.Thus, in this paper, we collected higher temporal resolution data that could reflect human activities, that is, PMIn the winter from 2019 to 2020, COVID-19 broke out in China. On January 23 of 2020, Wuhan, as the first city where COVID-19 cases appeared, announced that the city would lockdown to stop virus transmission. People in this city were not allowed to leave and advocated to stay at home. With the spread of the virus, other cities in China have gradually taken lockdown measures. Under this measure, human activities were greatly restricted, including the slowdown in the production of enterprises and the significant reduction in the travel of residents. This special period provides an unprecedented case for studying the impact of human activities on air pollutants.The univariate local Moran's I test was needed to assess spatial autocorrelation before we detected local agglomeration. The univariate local Moran's I index has been proven to be an effective tool for distinguishing the local spatial clusters, and it is calculated as shown in Equation :Yi is the value of the target variable (PM2.5 concentration) of the i-th city, Yj represents the value of the PM2.5 concentration in other cities (j≠i), and 2 is the variance of the target variable. ωij is the spatial weight displaying the neighboring relations among the geographical units. In this research, we used GeoDa software to obtain the LISA for cities' PM2.5 concentrations before and after the COVID-19 outbreak.Where 2.5 concentration may accompany spatial heterogeneity  denotes the i-th city's geographical position  and time location (ti). βk represent the coefficients of space-time observation sample i, which can be explained as the percentage of dependent variable (Y) change with 1% variation of the independent variable (X). εi is the random error term.Where k for variable k and spatiotemporal location i is examined by the following equation:βW is the spatiotemporal weight matrix estimated by the temporal and spatial distances and the decay functions. Therefore, the spatiotemporal weight for independent variable needs to be calculated before conducting this model analysis. The spatiotemporal distance dST could be split into spatial distance (dS) and temporal distance (dT), as shown in Equation  can be obtained by using Equation ] was used to find the optimal spatiotemporal bandwidth (Where andwidth .i(b) denotes the predicted value from the GTWR model, and the function is the sum of squared errors.Where ŷx is shown in Equation . To ensure the continuity and reliability of the original data, we conducted strict data quality control on the hourly PM2.5 concentration data before analysis according to the provisions on the validity of air pollutant concentration data (GB 3095-2012) (https://www.mee.gov.cn/), and we removed abnormal data. The daily average of each site and then averaged these daily averages of sites in each city was calculated as the daily PM2.5 concentration of the city. Based on the data pre-processing, 332 cities were selected in this study to discuss regional changes in PM2.5 concentrations.Hourly observations of the PM2.5 concentrations at 1,672 monitoring sites in our study period were collected from the real-time monitoring data system of the China National Environmental Monitoring Center (CNEMC) , temperature , relative humidity , and wind speed . In this work, rh was calculated by the dewpoint temperature (d2m) and temperature (t2m) based on the Clapeyron-Clausius equation. ws is the square root of the sum of the squares of the eastward component of the 10 m wind (u10) and the northward component of the 10 m wind (v10). The reanalysis dataset (ERA5-Land hourly data) for these data was obtained from the European Centre for Medium-Range Weather Forecasts (ECWMF) (https://www.ecmwf.int/). This reanalysis dataset is hourly 0.1 × 0.1 grid data. The city-scale meteorological data processing method is similar to that used for the PM2.5 concentrations.Four essential meteorological factors were selected to report the natural impacts on PMhttps://qianxi.baidu.com/). Therefore, we applied this index to indirectly reflect the impact of human travel and lifestyle changes on PM2.5 concentration . After the Spring Festival in 2020, under the strict implementation of epidemic control measures, the Chinese government announced that some essential enterprises could resume production with restrictions. We thus collected the daily pollutant operating vent numbers of more than 9,000 key polluting enterprises in China from January to February 2020 (http://www.ipe.org.cn/). These enterprises usually pose great environmental risks, such as a great quantity these air pollutants, and they are therefore screened by the environmental protection department of the local government based on the environmental quality improvement requirements of their respective administrative areas and other specified conditions , which could reflect the dynamic change in industrial pollution intensity. The numbers of daily pollutant operating vents of key polluting enterprises in the power sector (VOP) were used to report the variation in energy consumption. The pollutant operating vents were the sum of the vent numbers of dust, nitrogen oxide (NOx) and sulphur dioxide  on Jan 8, rapidly returned to 60 μg/m3, and continued to fluctuate upward. In contrast, as cities in China gradually locked down, the daily average national PM2.5 concentrations showed a clear trend of first increasing and then decreasing, with the value declining to 30.9 μg/m3 on February 14. This tendency was consistent with the findings of Wang et al. , the North China Plain , and the Tarim Basin (Xinjiang Province). Other areas with high PM2.5 concentrations (50–75μg/m3) were distributed around the regions with the highest concentrations. However, after cities gradually locked down, 84.3% of cities experienced a decline in PM2.5 concentration. The number of cities characterized by PM2.5 concentrations higher than 75 μg/m3 was significantly reduced, decreasing by 34.2%. Similar to the period before measures were taken, cities with high PM2.5 concentrations were concentrated in Northeast China, the North China Plain and Xinjiang Province. Moreover, haze pollution was improved in 30 cities, with PM2.5 concentrations <37.5 μg/m3. These cities were mainly distributed in the southern coastal areas.The daily average PMeristics . In the 2.5 concentration. During our study period, cities' daily PM2.5 concentrations presented an appearance of regional convergence, mainly marked by a high-high group and a low-low group. When human activities were not limited, the high-high group occurred in Northeast China, the North China Plain, and the Tarim Basin, which indicated that the PM2.5 pollution in these regions was more serious and that the positive local spatial autocorrelation of PM2.5 existed in these cities. Cities with low PM2.5 concentrations were mainly distributed in Xizang, Qinghai, Sichuan, Yunnan, Guizhou, and Fujian Provinces, suggesting that the haze pollution in these areas was relatively light. The local spatial autocorrelation of haze pollution also existed in these places but the correlation was weak. However, after lockdown measures were taken, the club-convergence phenomenon became more obvious, with the high-high cluster in Northeast China shifting to the south. The regions characterized by the low-low cluster in Northwest China shrank, while the regions in Southwest China expanded to Guangdong Province.We further conducted a local Moran's I test  to identR2 value was 0.403, the adjusted R2 was 0.402, the bandwidth value was 0.115, the sigma value was 0.259, the CV value was 808.202 and the spatiotemporal distance ratio was 1.000.The GTWR model was employed to calculated the influence of selected factors on cities' haze pollution in this study, and the parameters of the model are displayed in 2.5 concentrations in most cities were promoted. In contrast, the coefficients of rh, tem and ws were left-distributed, and the peaks emerged at approximately−0.08,−1.25, and−0.25, respectively, revealing that the increase in rh, tem and wind would restrain the raise of PM2.5 concentration in most cities during our study period.From the Kernel distribution of coefficients of different variables , we can 2.5 concentrations, that the increased sp in the south aggravated the PM2.5 concentrations, with Fangchengganng (0.609), Haikou (0.608) and Chongzuo (0.608) ranking among the top three cities. This is mainly because increased sp hindered the diffusion of pollutants in the vertical direction . Decreases of PM2.5 concentration in these cities was resulted from specific meteorological conditions formed by sp and other meteorological factors (The surface pressure (sp) coefficients for the 332 Chinese cites during our study period  showed airection . However factors .2.5 concentrations in southeast and middle regions of the country, which revealed that the increased temperature in these areas would bring with a reduction in PM2.5 levels. Among the cities, Wenzhou was found to preserve the most significant negative correlation between temperature and PM2.5 concentration, a related coefficient being−2.289. As higher surface temperature would intensify atmospheric convection, PM2.5 concentration in these cities reduced. In contrast, positive correlations between temperature and PM2.5 concentration was exhibited in north regions. The temperature in the Ali exerted the most positive impact on the PM2.5 concentration, with the correlation coefficient being 7.455. The winter temperature in northern cities was low , Aksu (0.577), and Bortala Mongolia (0.572), among others. In the environment with high rh, water vapor in the air was easy to condense water-droplets, which would lead to the growth of moisture absorption of particles and aggravate haze pollution  coefficients , we can ollution . Zhousha2.5 concentrations were not significant in the eastern and southern regions during our study period. Among the cities with a significant relation, the ws coefficients of several cities located in West and Northeast China were positive, but the coefficients were found to be negative in other cities. Specifically, the wind speed in Shigatse had the greatest promotion of haze pollution, with a coefficient of 0.121. The lowest ws coefficient emerged in Yingkou (- 0.057). As one of the important functions of wind was to transport air pollutants, a high wind speed contributed to the air pollutants' dilution and diffusion process, resulting in decreased regional PM2.5 concentrations  coefficients  showed ttrations .2.5 concentrations . Correlation coefficients in the southwestern and south-eastern coastal cities were less than zero, suggesting that raise of the VOI were accompanied by the decline of PM2.5 concentrations. Cities in northern China, the PM2.5 concentration was shown increase with grow in VOI. The VOI in Diqing Tibetan played a particularly substantial role in decreasing the PM2.5 concentration, with the coefficient of−0.075, then, the Nujiang Lisu Nationality Autonomous region (- 0.074), Lijiang (- 0.074), and Dali (- 0.071). The VOI in Turpan exhibited the greatest promoting influence on the PM2.5 concentration, having the coefficient of 0.230.As seen from 2.5 concentrations than in other cities. Hulun Buir (-0.070) had the lowest coefficient, followed by Heihe (-0.056) and Qiqihar (-0.053). The significance of the positive influence of VOP on PM2.5 concentrations in Shigatse, Lhasa, and Nagqu ranked as the top three among cities, with coefficients of 0.549, 0.443, and 0.419, respectively.Contrary to the VOI results, the coefficient of VOP demonstrated an upward trend as cities shifted from the northeast to the southwest of China . In nort2.5 concentration was obvious. The decrease of PM2.5 concentration in NCP was mainly caused by the increase of temperature and wind speed under the meteorological conditions at that time. Compared with other natural factors, the rise of temperature was the main reason for the mitigation of haze pollution in the YRD. In most cities in NEC, the reduction of relative humidity was the most important natural factor to reduce haze pollution. In addition, the main natural factor causing the decrease of PM2.5 concentration in some cities in Xinjiang was the increase of wind speed.During the study period, the areas with serious haze pollution in China were mainly concentrated in the North China Plain (NCP), the north of the Yangtze River Delta (YRD), Northeast China (NEC) and some cities in Xinjiang. The specific area is shown in 2.5 concentration during our study period. The intensity of trip activities (ITAs) presented a positive effect on haze pollution, denoting that the limitation of travel intensity would generally reduce the PM2.5 concentration  were mostly situated in Northeast China, the North China Plain and the Tarim Basin. As the problem of haze pollution was suppressed, the spatial aggregation of PM2.5 concentration was more prominent. The surface pressure coefficients for Chinese cities increased from the north to the southwest of China. The coefficient of temperature exhibited negative correlations with PM2.5 concentrations in southeast and middle cities; inversely, positive relationships were found in the northeast and northwest regions. Cities with higher relevance of PM2.5 concentrations with relative humidity were mainly situated in Northwest China. Regions in western and north-eastern China witnessed a significant positive influence of wind speed on PM2.5 concentrations. Among the three anthropogenic factors we considered, the ITAs exhibited significant positive influence on haze pollution, and the correlation maintained a high intensity during the study period. The VOI coefficients in northern cities were positive, while those in south-western and south-eastern coastal cities shown significantly negative. In addition, the VOP coefficients demonstrated an uptrend as region shifted from the northeast to the southwest of the country. Based on the spatiotemporal heterogeneity of coefficients for each factor, policy for controlling haze pollution need to be pointed out by considering the distinct meteorological and human activities at regional scales. Reasonable restrictions on people's travel intensity can effectively prevent haze pollution. In winter, cities in the north could focus on eliminating the operating vents of enterprises in the industrial sector with emission levels exceeding the regional standard limits, while cities in the west and south could pay more attention to the reduction of unnecessary pollutant emissions of polluting enterprises in the power sector.After the strict implementation of epidemic control measures gradually taken in Chinese cities, 84.3% of cities' PMhttps://qianxi.baidu.com/. The numbers of daily pollutant operating vents of key polluting enterprises can be found here: http://www.ipe.org.cn/. Meteorological data can be found here: https://www.ecmwf.int/, and observations of PM2.5 concentrations can be found here: https://air.cnemc.cn:18007/.Publicly available datasets were analyzed in this study. Data of intensity of trip activities can be found here: LY: formal analysis, data curation, writing-original draft, and writing-review and editing. SH, SY and ZW: writing-review and editing. CH: writing-review and editing and resources. JH: data curation. ZY: data curation and validation. BC: resources. YW: writing-original draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Under a low-input production system, local cattle have extraordinary adaptability to a variety of environments. However, due to various reasons, Native Altay white-headed cattle selection and breeding have not received enough attention. In addition, a large number of excellent foreign cattle varieties have been introduced, and unplanned hybridization has been carried out. The number of pure Altay white-headed cattle has been decreasing, with the breed even facing the danger of extinction. The genetic diversity index is of great significance for evaluating population structure and formulating conservation strategies. The genetic variation and population structure of Altay genetic resources were studied using 100 k SNP genotyping data from Altay white-headed cattle. Our results show that the genetic structure of the Altay white-headed cattle population is different, but the level of genetic differentiation is low, and the genetic diversity is low. In conclusion, our findings provide genetic information about the Altay white-headed cattle population, which can be used for future conservation and breeding research.Understanding the genetic basis of native cattle populations that have adapted to the local environment is of great significance for formulating appropriate strategies and programs for genetic improvement and protection. Therefore, it is necessary to understand the genetic diversity and population structure of Altay white-headed cattle so as to meet the current production needs under various environments, carry out continuous genetic improvement, and promote rapid adaptation to changing environments and breeding objectives. A total of 46 individual samples of endangered Xinjiang Altay white-headed cattle were collected in this study, including nine bulls and 37 cows. To collect genotype data, 100 k SNP markers were used, and then studies of genetic diversity, genetic structure, inbreeding degree, and family analysis were carried out. A total of 101,220 SNP loci were detected, and the genotype detection rate for individuals was ≥90%. There were 85,993 SNP loci that passed quality control, of which 93.5% were polymorphic. The average effective allele number was 0.036, the Polymorphism Information Content was 0.304 and the minimum allele frequency was 0.309, the average observed heterozygosity was 0.413, and the average expected heterozygosity was 0.403. The average genetic distance of Idengtical By State (IBS) was 0.3090, there were 461 ROH (genome-length homozygous fragments), 76.1% of which were between 1 and 5 MB in length, and the average inbreeding coefficient was 0.016. The 46 Altay white-headed cattle were divided into their families, and the individual numbers of each family were obviously different. To sum up, the Altay white-headed cattle conservation population had low heterozygosity, a high inbreeding degree, few families, and large differences in the number of individuals in each family, which can easily cause a loss of genetic diversity. In the follow-up seed conservation process, seed selection and matching should be carried out according to the divided families to ensure the long-term protection of Altay white-headed cattle genetic resources. China has a vast territory with complex and diverse terrain, giving birth to many cattle breeds with local characteristics. For example, Xinjiang Altay white-headed cattle are favored by the majority of farmers and herdsmen in Xinjiang due to their excellent characteristics such as early maturity, strong reproductive ability, fast growth and development, high cold resistance, high temperature resistance, rough feeding resistance, good adaptability, and strong disease resistance. They also have good production capacity, tender meat, a high milk fat rate, a beautiful shape, and a gentle character, with ancient and primitive strains .Worldwide, the cattle subfamily mainly includes six genera, 13 cattle species, and nearly 800 varieties. Due to the different times of origin and differentiation, the living environment and geographical distribution of different cattle breeds are also quite different. Altay white-headed cattle are an important breed of livestock in Altay. They make potential contributions to rural livelihoods, organic agriculture, and sociocultural and historical events. The number of cattle in the region has reached 733,000, with 525,100 in stock. The main cattle varieties are Xinjiang brown cattle, Holstein, Simmental, white-headed cattle, and Kazakh cattle . As HemuGenetic diversity is an important component of biodiversity. Generalized genetic diversity refers to the sum of various pieces of genetic information carried by organisms on the earth. In the narrow sense, genetic diversity is mainly the variation in intraspecific genes, including the genetic variation between different populations and within the same population . GeneticThe CWE9600 Maghead Blood DNA Kit from China Jiangsu Kangwei Century Biotechnology Co., Ltd., Taizhou, China, red blood cell lysate (RT122), 6× Loading Buffer, molecular weight marker, fast agar gel DNA recovery kit, Hot Start Taq polymerase, agarose, and anhydrous ethanol were purchased from China Beijing Tiangen Biochemical Technology Co., Ltd., Beijing, China.Blood samples from the jugular vein of 46 Altay white-headed male and female cattle, including 37 cows and nine bulls, were collected in the Hemukanas Mongolian Nationality Township, Altay City, Xinjiang Province, and stored in a refrigerator at −80 ℃.The magnetic bead method was used to extract DNA. Based on the principle of specific adsorption of DNA by silicon-based magnetic beads, the cwe9600 magbead blood DNA kit was used to release the blood genome through enzymatic hydrolysis, bind DNA molecules in a specific high-efficiency lysis buffer system, and change the environmental ionic strength so as to realize the isolation and purification of genomic DNA.After extraction, a 2% agarose gel was used for electrophoresis to detect the quality, a gel imager was used to observe the size and brightness of genomic DNA fragments, a Qubit was used to accurately quantify the DNA concentration, a Nanodrop was used to detect the purity of DNA samples (OD260/OD280 = 1.7–2.1), and photos were taken for recording.To extract qualified gDNA (concentration ≥ 50 NG/UL), first the whole genome of all samples was amplified . Afterwards, the gDNA was incubated at 37 °C for 20–24 h, and then it was fragmented, precipitated, and resuspended in hybridization buffer. The resuspended DNA fragments were added to the chip, hybridized, and incubated at 48 °C for 16–24 h. After hybridization, the non-specific binding DNA was removed by washing, and the specific binding sites were left for single-base extension. After staining, the data were scanned and read with the Illumina iSCAN reader for data analysis.Genetic diversity analysis includes the effective population size (Ne), which refers to the ideal population content with the same gene frequency variance or the same inbreeding coefficient increment (heterozygosity decay rate) as the actual population . It is uPlink1.9b software was used to analyze runs of homozygosity (ROHs). First, Plink (v1.90) was used to calculate the ROH length of each sample. The inbreeding coefficient based on ROHs was calculated by calculating the proportion of the total length of ROH fragments in an individual to the total length of the autosomal genome . TherefoThe genomic relationship G matrix was constructed using the genome-wide marker information, and the G matrix molecular kinship analysis was carried out using GCTA (V1.94) software. A heat map was drawn to show the genomic kinship among individuals . The PliCluster analysis is a method of generating a relatively simple class structure from a group of complex data and classifying groups according to the degree of correlation or similarity between different individuals . We usedThe concentration of the 46 Altay white-headed cattle DNA samples was more than 100 ng/μ 50. The Od260 nm/od280 nm was 1.7~2.0. The genomic DNA samples were of good quality, without protein pollution and degradation In order to ensure the accuracy of the analysis, Plink (v1.90) software was first used to check the duplicate samples. The criterion for judging the duplicate samples was as follows: when the DST between samples was ≥0.99, the samples were judged as duplicate samples. DST is the probability that two individuals reflect homomorphism at the genomic level. Two pairs of duplicate samples were found in this analysis. We reserved the samples with a high detection rate for analysis.Plink (v1.90) software was used for the quality control of SNP loci, and only the loci with the best typing quality were reserved for subsequent analysis. The results of the genetic diversity analysis are shown in A total of 461 ROH fragments were detected in the conservation population of 46 Altay white-headed cattle, with the largest number of ROHs being between 1 and 5 MB in length, accounting for 76.1%, as shown in The number of individuals with a total length of ROHs between 0 and 50 MB was the largest, with 32 heads in total, as shown in In this experiment, a total of 101,220 SNP loci were detected in 46 samples, and 85,993 SNP loci after quality control were used for principal component analysis. It can be seen from The genetic distance of IBS among the 46 Altay white-headed cattle was between 0.1927 and 0.3451, with an average of 0.3090. The results of the IBS distance matrix of the Altay white-headed cattle conservation population are shown in In view of the importance of bulls to the whole conservation population, we extracted samples of bulls and conducted cluster analysis separately to judge their kinship. The results are shown in Combined with the results of the genomic kinship analysis and cluster analysis, the existing bull samples were divided into one family. The standard for clustering is that the genomic kinship coefficient between bulls is greater than or equal to 0.1. According to the kinship between the existing cows and bulls, different families were established. In addition, it was found that 30 cows were distant from the detected bulls. Therefore, they were classified into the “other” category  may be the result of special mixing between different cattle breeds. Thus far, the Altay white-headed cattle population has unique genetic resources and untapped opportunities, and measures need to be taken to protect and utilize this population sustainably.The average inbreeding coefficient of this group was 0.016. In order to reduce the inbreeding increment in the population, it is recommended not to breed bulls and cows of the same family. The genetic relationship coefficient between the 30 cows in the “other” category in the family group construction results and all the bulls was less than 0.1, meaning that these cows can be mated with any bull. After the genetic coefficient is confirmed, breeding can be carried out. An effective way to reduce the inbreeding coefficient is to expand the population content and carry out appropriate selection while strengthening management to avoid incomplete or incorrect pedigree records caused by inbreeding. Breeding bulls with large genetic distances can be selected to increase the effective population size. This study also found that the Altay white-headed cattle conservation group was only composed of three families, and the number of male and female cattle in the three families was quite different, with an unbalanced family structure. Therefore, it is necessary to introduce new lineages from other regions, expand the core group, and implement reasonable selection to avoid inbreeding and maintain the genetic diversity of the Altay white-headed cattle conservation population.In this study, we found that the genetic diversity of the Altay white-headed cattle population was very low through the detection and analysis of a 100 k gene chip, especially according to the genetic coefficient, where there was only one family left for the bulls. The genetic cluster analysis of Altay white-headed cattle proved the uniqueness of Altay white-headed cattle in molecular genetic structure. This helps to understand the endangered situation of this population, which is conducive to our emergency protection of endangered species. Once the genetic distance between bulls and cows is known, first-generation bulls and cows selected for mating should have a relatively large genetic distance. In each generation, the genetic distance between individuals is determined to ensure that bulls and cows with the farthest genetic distance can produce offspring by mating, so as to ensure that the genetic diversity of the population is maintained at the maximum level. The selection accuracy of bulls can be improved through the combination of multiple breeding methods. This lays the foundation for the improvement of Altay white-headed cattle breeding.