Modern research shows that Haima Duobian pill (HDP) can relieve the kidney yang deficiency syndrome (KYDS), but the mechanism is still unclear. The aim of this work was to study the effects of HDP in a rat model of KYDS.  The network pharmacology methods were used to predict the therapeutic effects of Haima Duobian pill. Adenine was used to establish the rat model of kidney yang deficiency syndrome. The general physical signs of rats were observed after different doses of Haima Duobian pill (HDP) were given. Serum cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), estradiol (E2), and gonadotropin-releasing hormone (GnRH) levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. Then, the histopathologic changes and sperm activity were detected.  HDP could improve the general signs of kidney yang deficiency syndrome rats. After the rats were treated with HDP, the expression of cGMP and E2 was significantly inhibited and the expression of cAMP and T was significantly increased. The pathological damage of testis, epididymis, and seminal vesicle was alleviated, and the sperm activity was improved.  For adenine-induced kidney yang deficiency syndrome in rats, HDP had a significant therapeutic effect. Kidney yang deficiency syndrome (KYDS), a diagnostic pattern in Chinese medicine, has clinical features similar to those of the glucocorticoid withdrawal syndrome . It is aHaima Duobian pill (HDP) is a classic traditional Chinese medicine (TCM) formula for the treatment of KYDS and has been widely used to recuperate KYDS clinically for hundreds of years in China. There are few studies on the mechanism of HDP action. Based on the HPG axis, this study will explore the effect of HDP on spermatogenesis in rats with KYDS.The Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to screen the bioactive compounds. The ADME parameter-based virtual screening of the compounds was utilized to further identify compounds using an oral bioavailability (OB) threshold OB ≥30% and a drug-likeness (DL) threshold DL ≥0.18.TCMSP was used to search for potential targets associated with active components.https://omim.org/) and GeneCards (http://www.genecards.org/) databases and got 1122 disease-related targets.With “dysfunction of hypothalamic-pituitary-gonadal axis” as the key word, the research team searched OMIM (https://https://string-db.org/) was used to visualize the information.The effective targets of Haima Duobian pill and the related targets of the dysfunction of hypothalamic-pituitary-gonadal axis were analyzed with R programming language. A total of 46 intersection targets were obtained. The online STRING database , with the column temperature at 30°C. The elution flow rate was 1.0 mL/min with a mobile phase gradient of A − B , which was varied as follows: 0–30 min, 51% A; 30–60 min, 51%–80% A; 60–70 min, 80%–51% A; 70–80 min, 51% A. Schisandrol A was detected at UV 210 nm. The HPLC chromatogram of Haima Duobian pill is shown in The quality control of Haima Duobian pill was analyzed using high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection . The powder of Haima Duobian pill was extracted with water saturated ethyl acetate. The extract was evaporated to dryness. The residue was dissolved in methanol. The solution was filtered, and then 10 SD male rats, weighing 200–300 g, were provided by Chengdu Dashuo Experimental Animal Co., Ltd., production permit: SCXK (Chuan) 2015-030. The rats were fed adaptively for 3 days, during which they could drink freely. Then, the rats were divided into 6 groups (12 rats in each group): normal group (NG), model group (MG), high dose group (HDG), middle dose group (MDG), low dose group (LDG), and positive group . The experiment was completed in the Pharmacology Laboratory of Traditional Chinese Medicine of Chengdu University of Traditional Chinese Medicine. The laboratory is the Research Laboratory of Traditional Chinese Medicine of the State Administration of Traditional Chinese Medicine (Level III), Certificate Registration No. TCM-2009-315.Hippocampus), Gejie (Gekko gecko), Jiucaizi (Allii Tuberosi Semen), Suoyang (Cynomorium songaricum Rupr.), Lurong (Cornu cervi pantotrichum), Buguzhi , Xiaohuixiang (Foeniculi Fructus), Tusizi (Cuscutae Semen), Shayuanzi , Shanzhuyu , Baizhu , Duzhong (Eucommiae Cortex), Hongshen (Ginseng Radix et Rhizoma Rubra), Mudingxiang (Fruit of Caryophylli Flos), Niuxi (Achyranthis Bidentatae Radix), Fuling (Poria cocos (Schw.) Wolf.), Shanyao (Rhizoma Dioscoreae), Huangqi (Hedysarum multijugum Maxim.), Danggui (Angelicae Sinensis Radix), Longgu, Gancao (Licorice), Rougui (Cinnamomi Cortex), Quenao (Brain marrow of Passer montanus (Linnaeus)), Wuweizi (Schisandrae Sphenantherae Fructus), Gouqizi (Lycii Fructus), Goubian (Dog whip), Lvbian (Donkey whip), Niubian (Bullwhip), Diaobian (Mink whip), Shudihuang (Rehmanniae Radix Praeparata), Fuzi , Roucongrong (Cistanches Herba), Bajitian , Yinyanghuo (Epimedii Herba). Wuzi Yanzong pill , Schisandrol A , Adenine , Rat LH (Luteinizing Hormone) ELISA Kit (Lot: 3XNQ4LLQH7), Rat FSH (follicle-stimulating hormone) ELISA Kit (Lot: UT7DCSJV5Y), cAMP (cyclic adenosine monophosphate) ELISA Kit (Lot: UA4SG5RCJW), GnRH (gonadotropin-releasing hormone) ELISA Kit (Lot: 1TP7S3C8HH), cGMP (cyclic GMP) ELISA Kit (Lot: K6LFPAPG7N), T (Testosterone) ELISA Kit (Lot: K4P1SC1WZS), and Rat/Porcine E2 (Estradiol) ELISA Kit (Lot: CL8NR4F5FH). All of the above kits were purchased from Wuhan Yilairuite Biotechnology Co., Ltd.Haima Duobian pill  was used. Haima Duobian pill is composed of Haima  Organ Index Test. After intraperitoneal injections of sodium pentobarbital, the kidney, testis, epididymis, and seminal vesicle gland of male rats were extracted and weighed, and the organ index of each organ or tissue was calculated by the following formula: organ index = organ weight/body weight × 100%.(2) Measurement of Serum Cytokines. After intraperitoneal injections of sodium pentobarbital, 5 ml of abdominal aortic blood was taken from each rat (rats were able to drink freely 12 hours before taking blood). After 2 hours, the blood samples were centrifuged at 3000 r/min for 10 min, and the serum was taken. The contents of cAMP, cGMP, GnRH, LH, FSH, T, and E2 in the serum were determined by ELISA Kit.(3) Sperm Activity Test. After intraperitoneal injections of sodium pentobarbital, the epididymis of each rat was taken. It was put into 8 ml normal saline and cut to pieces to make its distribution uniform. The suspension was put in a 37°C water bath for 10 min and filtered, and normal saline was added to 8 ml and prepared as sperm suspension. One drop of sperm suspension was taken and observed under microscope. According to the WHO manual, sperm motility was divided into four grades: A (fast forward movement), B (slow forward movement), C (nonforward movement), and D (no movement). The ratio of A + B grade sperm was calculated [lculated , 6.(4) Histopathological Observation. The organs were fixed and observed under microscope after HE staining.(5) Statistical Analysis. SPSS 19.0 was used to analyze the data. Gaussian distribution of each data was evaluated using the Shapiro–Wilk normality test. Differences among groups were analyzed using one-way analysis of variance (ANOVA). Normally distributed measurement data were expressed as the mean ± standard deviation (SD). P < 0.05 showed that the difference was statistically significant.Homo sapiens. Setting the medium confidence to 0.400 and hiding the disconnected nodes in the network, we could obtain the protein-protein interaction information . Compared with the model group, the weight growth rate of the low dose group and the positive group increased significantly (P < 0.05). After that, we analyzed the body temperature of each group of rats, as shown in P < 0.01). Compared with the model group, the body temperature of high dose, middle dose, low dose, and positive groups was significantly higher (P < 0.01 or P < 0.05).First, we determined the differences in weight growth rate among the groups. The results are shown in P < 0.01). It may be due to adenine deposition. Compared with the model group, the renal index of the high dose group of HDP was lower, but the difference was not statistically significant (P > 0.05). Compared with the normal group, the testis index of the model group was lower, and the difference was statistically significant (P < 0.05). Compared with the model group, the testis index of the high dose group, the middle dose group, and the low dose group of HDP was higher, and the difference was statistically significant (P < 0.01). The testis index of the positive group was higher than that of the model group (P < 0.05). Compared with the normal group, the epididymis index of the model group was lower, and the difference was statistically significant (P < 0.05). Compared with the model group, the epididymis index of the high dose group, the middle dose group, and the low dose group of HDP was higher, and the difference was statistically significant (P < 0.01). The epididymis index of the positive group was also higher than that of the model group, and the difference was statistically significant (P < 0.05). Compared with the normal group, the seminal vesicle index of the model group was lower, and the difference was statistically significant (P < 0.05). Compared with the model group, the seminal vesicle index of the high dose group of HDP was higher, but the difference was not statistically significant (P > 0.05). These results indicate that HDP has the tendency to improve the atrophy of gonadal axis target organs. The results are shown in Compared with the normal group, the kidney index of the model group was significantly higher (P < 0.05). Compared with the model group, the serum cAMP of the high dose and middle dose groups of HDP and the positive group was higher, and the difference was statistically significant (P < 0.01). Compared with the normal group, the cGMP content in the serum of the model group was higher, and the difference was statistically significant (P < 0.05). Compared with the model group, the cGMP content in the serum of the high dose group, the middle dose group, and the positive group was lower, and the difference was statistically significant (P < 0.05). Compared with the normal group, the cAMP/cGMP in the serum of the model group was lower, and the difference was statistically significant (P < 0.05). Compared with the model group, the serum cAMP/cGMP in the high dose group, the middle dose group, and the low dose group of HDP was higher, with statistical significance (P < 0.01 or P < 0.05). The serum cAMP/cGMP in the positive group was higher than that in the model group, with statistical significance (P < 0.01). It can be concluded that HDP can regulate the levels of cAMP and cGMP in the serum of rats with KYDS. The results are shown in Compared with the normal group, the serum cAMP of the model group was lower, and the difference was statistically significant (P < 0.01); compared with the model group, the difference of serum LH level of the other groups had no statistical significance (P > 0.05). Compared with the normal group, the level of FSH in the serum of the model group was higher, and the difference was statistically significant (P < 0.05). Compared with the model group, there was no significant difference in serum FSH level of the other administration groups (P > 0.05). Compared with the normal group, the content of T in the serum of the model group was lower, and the difference was statistically significant (P < 0.01). Compared with the model group, the content of T in the serum of the high dose group, the middle dose group, and the positive group was higher, and the difference was statistically significant (P < 0.01 or P < 0.05). Compared with the normal group, the serum E2 content of the model group was higher, and the difference was statistically significant (P < 0.01). Compared with the model group, the serum E2 content of the high dose group and positive group was lower, and the difference was statistically significant (P < 0.05). Compared with the normal group, the content of GnRH in the serum of the model group was higher, and the difference was statistically significant (P < 0.01). Compared with the model group, there was no significant difference in the serum GnRH level of administration group (P > 0.05). These results indicate that HDP can regulate the secretion of cytokines, increase the level of T, and improve the clinical symptoms of KYDS. The results are shown in Compared with the normal group, the serum LH level of the model group was higher, and the difference was statistically significant . Compared with the model group, the ratio of A + B grade sperm in the high dose group and positive group increased significantly . The results are shown in Compared with the normal group, the ratio of The boundary between renal cortex and medulla was clear in normal group. The number of renal tubules was abundant, and the epithelial cells were normal. The renal volume of model group and other administration groups increased. A large number of adenine crystals were seen, some of which were encapsulated by macrophages and multinucleated macrophages, forming granuloma. More renal tubules were atrophied, the lumen became narrower, and the volume of epithelial cells decreased. The basement membrane of renal tubules was thickened. There were more connective tissue hyperplasia and inflammatory cell infiltration in renal tubules. The results are shown in In the normal group, the shape of seminiferous tubules was regular. The spermatogenic cells and Sertoli cells in tubules were closely arranged and abundant, and long fusiform spermatozoa could be seen. No other obvious abnormality was found in the tissues. In the model group, the arrangement of spermatids in the seminiferous tubules was disordered. The connective tissue between the seminiferous tubules was increased. The number of interstitial cells was significantly reduced. The number of spermatozoa was greatly reduced, and the seminiferous cells were exfoliated in some tubules. In the high dose group, the shape of seminiferous tubules was regular, and the gap was widened. The spermatogenic cells were arranged closely and abundant. Long fusiform spermatozoa could be seen, and no other obvious abnormality was found in the tissues. The shape of seminiferous tubules in the middle dose group was regular, and the gap was widened. A small number of seminiferous tubules had a large number of decreased spermatocytes, and no other obvious abnormality was found in the tissues. In the low dose group, the shape of seminiferous tubules was regular, and the gap was widened. The spermatogenic cells were closely arranged, and long fusiform spermatozoa could be seen. The number of spermatozoa in some seminiferous tubules was significantly reduced, and no other obvious abnormality was found in the tissues. In the positive group, the shape of seminiferous tubules was regular, and the gap was widened. The spermatogenic cells were arranged closely and abundant, and there were long fusiform spermatozoa in the tubules. No other obvious abnormality was found in the tissues. The results are shown in In the normal group, the epithelial cells of epididymis were normal in morphology, with clear boundary and no other obvious abnormality. In the model group, the epididymal epithelial cells were necrosis, and the nuclei were pyknosis, hyperchromatism, or fragmentation. The lumen was filled with sperm and eosinophilic protein fluid. Interstitial connective tissue was loose, and a small number of inflammatory cells were infiltrated. In the high dose group, the epithelial cells of epididymis were normal in morphology, with clear boundary and no other obvious abnormality. In the middle dose group, some epididymal epithelial cells were swollen. The cytoplasm was loose and light stained. No other obvious abnormality was found. In the low dose group, the epididymal epithelial cells swelled and the number of sperm in some lumens decreased significantly. In the positive group, the morphology of epididymal epithelial cells was normal, the boundary was clear, and the interstitial connective tissue was loose. There was no other obvious abnormality. The results are shown in In the normal group, the structure of each layer of seminal vesicle was clear. The mucosal layer and muscular layer were closely arranged, and a series of folds could be seen in the mucosal layer. The epithelium of the mucosal layer was a single columnar epithelium, and the epithelial cells were closely arranged with normal morphology. No other obvious abnormality was found in the tissue. The seminal vesicles of other groups were dilated, and no other obvious tissue abnormality was found. The results are shown in Kidney yang deficiency syndrome (KYDS), a metabolic disease caused by a neuroendocrine disorder, was recorded first in an early systematic and theoretical monograph existing in China, “Neijing” . The theReproduction is controlled by the hypothalamic-pituitary-gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) neurons can produce GnRH and stimulate the biosynthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). GnRH is the main hypothalamic regulator of the release of gonadotropins. FSH and LH regulate the function of testis through their receptors in Sertoli and Leydig cells, respectively . LH, FSHPrevious studies have shown that the kidney yang deficiency syndrome model constructed by intragastric administration of adenine shows that the renal tubules of animals are seriously dilated, and the epithelial cells of renal tubules show edema and inflammatory cell infiltration. The diameter of most seminiferous tubules became thinner. The seminiferous epithelium became thinner. The arrangement of spermatogenic cells was disordered, and the number and level of spermatogenic cells decreased. The wall of epididymal tube is thickened, arranged loosely, the interstitial connective tissue is enlarged, the lumen is proliferated and atrophied, and the sperm in the lumen accumulates into a mass , 19, 20.The efficacy of HDP includes tonifying kidney, strengthening yang, adding essence, and increasing marrow. It can be used to treat qi and blood deficiency, sallow and emaciated face, nocturnal spermatorrhea, premature ejaculation, impotence, and sore waist and legs. In this experiment, the kidney yang deficiency syndrome (KYDS) was induced by intragastric administration of adenine. Based on the HPG axis, we explored the role of HDP in improving KYDS. From the above experimental results, HDP can improve the general signs of rats with KYDS. Through the detection of organ index, it was found that HDP could improve the atrophy trend of testis, epididymis, and seminal vesicle gland. According to enzyme-linked immunosorbent assays, we determined the serum cytokines of rats and found that HDP can regulate the endocrine disorder of rats. The sperm activity test showed that HDP could improve the sperm activity of rats with KYDS. Histopathological observation showed that HDP could alleviate the pathological damage of testis, epididymis, and seminal vesicle gland to a certain extent. Furthermore, HDP may improve KYDS through other mechanisms, including altered energy metabolism, lipid metabolism, gut microbiota metabolism, and biosynthesis of catecholamine, which is worthy of further study.Severe KYDS can lead to abnormal spermatogenesis and infertility. This may be closely related to the hypothalamic-pituitary-gonadal axis. HDP can improve KYDS based on hypothalamic-pituitary-gonadal axis. This study provides a basis for the effectiveness of HDP in the treatment of KYDS.


The objective of this study was to estimate the genetic parameters and trends for milk, fat, and protein yields in the first three lactations of Thai dairy cattle using a 3-trait,-3-lactation random regression test-day model.Data included 168,996, 63,388, and 27,145 test-day records from the first, second, and third lactations, respectively. Records were from 19,068 cows calving from 1993 to 2013 in 124 herds. (Co) variance components were estimated by Bayesian methods. Gibbs sampling was used to obtain posterior distributions. The model included herd-year-month of testing, breed group-season of calving-month in tested milk group, linear and quadratic age at calving as fixed effects, and random regression coefficients for additive genetic and permanent environmental effects, which were defined as modified constant, linear, quadratic, cubic and quartic Legendre coefficients.Average daily heritabilities ranged from 0.36 to 0.48 for milk, 0.33 to 0.44 for fat and 0.37 to 0.48 for protein yields; they were higher in the third lactation for all traits. Heritabilities of test-day milk and protein yields for selected days in milk were higher in the middle than at the beginning or end of lactation, whereas those for test-day fat yields were high at the beginning and end of lactation. Genetics correlations (305-d yield) among production yields within lactations (0.44 to 0.69) were higher than those across lactations (0.36 to 0.68). The largest genetic correlation was observed between the first and second lactation. The genetic trends of 305-d milk, fat and protein yields were 230 to 250, 25 to 29, and 30 to 35 kg per year, respectively.A random regression model seems to be a flexible and reliable procedure for the genetic evaluation of production yields. It can be used to perform breeding value estimation for national genetic evaluation in the Thai dairy cattle population. The breeding improvement program of dairy cattle in Thailand has been established for almost 60 years. It started from the establishment of an artificial insemination (AI) service in 1956. In the beginning, both live purebred dairy cattle and frozen semen were imported from temperate countries. The frozen semen was crossed with Thai native cattle to produce dairy crossbreeds. Nowadays, through selection and inter se mating, Thailand has tried to develop its own tropical dairy breed based on the Holstein Friesian (HF) and Thai native cattle. The HF was selected to be the predominant breed according to its size and milk ability, which is considered suitable to the local marketing system and socio-economic conditions of Thailand. At present, dairy crossbreds with ≥87.5% HF blood levels are the main population in Thailand.In 2018, the Thai dairy cattle population was reported to be 623,427 heads with 283,089 cows on 17,925 farms, with a raw milk production of around 3,000 tons per day [Performance records  and a progeny testing program, together with a semen production unit, were established and processed by the Department of Livestock Development (DLD) in 1969. With these activities, the first sire genetic evaluation in Thailand, using the herd-mate comparison method, was performed in 1985. However, the activities were only developed on a small scale with limited records. In 1990, the government of Thailand via the DLD, which plays the important role of developing a suitable breed for tropical conditions, approved a project named “Master Bull Project” to develop a full-scale progeny testing program. The project aims to promote a large-scale milk sampling and recording program throughout the country. The objective of the project is to run an effective progeny testing program to select the elite sires with inherited genetics suitable for tropical conditions. Up to the present, the genetic evaluation for production traits in dairy cattle has been used to test day (TD) records with test-day models (TDMs) instead of lactation models.Among various TDMs, a TD production with a random regression model (RR-TDM) has been proven to increase the accuracy of breeding value predictions. In addition, cows can be evaluated with any number of tests and the model can account for different genetic, permanent environmental (PE) and residual (R) variances over the course of lactation. This model has been widely implemented in the national genetic evaluations of dairy cows in many countries, and is being developed in many other countries, from fitting various functions to model additive genetic lactation curves, especially a multiple-trait, multiple-lactation TDM fitting random regression –4. LegenThe analysis of multiple-trait, multiple-lactation data has shown that genetic correlations among traits in different lactations are less than consistent , indicatIn Thailand, some TDM studies have used only single lactation TDMs or only one trait in a small data set ,9. HowevTherefore, the objective of this study was to present some results (genetic parameters and trends for milk production) from the application of multiple-trait, multiple-lactation random regression TDMs to a national genetic evaluation system for the production traits of a tropical dairy population, like in Thailand.The TD records of Thai dairy cows from the first three lactations, with calving between 1993 and 2013, were obtained from the dairy database of the Bureau of Biotechnology in Livestock Production (BBLP), DLD. To obtain data sets with a consistent size, the following records were included: age at calving was restricted to 18 to 48, 30 to 60, and 41 to 75 months for the first, second and third lactations respectively; an interval of between 5 and 35 days from parturition to the first TD; daily milk yields between 2 and 40 kg; at least 5 TD records per lactation; a minimum of 150 days in milk (DIM). Furthermore, all three traits  were required for each TD. All third lactation cows were required to have first and second lactation records. Likewise, the second lactation cows were required to have first lactation records. Additionally, the sires for the cows in the dataset were identified.After applying these criteria, a total of 168,996, 63,388, and 27,145 TD records  of the first, second and third lactations, respectively, measured in different calendar months within herds from 29,230 lactations, which were daughters of 1,116 sires belonging to 124 herds, were left to be analyzed. Ancestors of cows in the final data set were traced back in pedigree as far as the parents were known. Furthermore, the numbers of TD records for milk, fat and protein yields were not equal because fat and protein yields were missing in some TD due to technical reasons.The breed groups were classified into three groups using the percentage of Holstein Friesian (HF) blood level: <87.5% HF, 87.50% HF to 93.75% HF and >93.75% HF. Because dairy cattle in Thailand used up-grading system to increase milk yield and expected <87.5% HF group provide low milk yield, 87.50% HF to 93.75% HF group provide medium milk yield and >93.75% HF group provide high milk yield. The months of milk testing were divided into 12 months, as follows: January to December. Calving seasons were classified into three groups: winter (November to February), summer (March to June) and rainy season (July to October). Details of the final data file are given in Data were analyzed with a 3-trait, 3-lactation RR-TDM. The matrix notation of the model isy = a vector of milk, fat and protein yields; b = a vector of the fixed effects: herd-year-month of testing, breed-season of calving-month in tested milk group, and linear and quadratic age at calving; a = a vector of RR coefficients for animal genetic (AG) effect; p = a vector of RR coefficients for permanent environmental (PE) effect; e = a vector of residual (R) effects; Q = a matrix of five modified Legendre polynomials , as defined by Gengler et al [X, Z, and W = incidence matrices relating observations to various effects. The residual effects were taken to be independently distributed and with constant variance along DIM. Modeling of the herd-year-month of testing effect as a fixed effect across lactations was found to be advantageous in populations with small herd sizes [where er et al ; and X, rd sizes . Druet erd sizes  also fouA = the numerator relationship matrix; ⊗ = a Kronecker product function; G0 and P0 = 45×45 for (co)variance matrices of RR coefficients for AG and PE effects, respectively; I = an identity matrix (the number of cows with records); and R = 9×9 for diagonal matrix of residual variance matrices corresponding to each trait with elements on lactation n assumed to be constant throughout the lactation.where Estimates of (co)variance components for 3-trait, 3-lactation analyses were estimated using a Bayesian method via Gibbs sampling. Computations were performed using the software GIBBS2F90 . A unifoα̂) in the TDMs were used to form estimated breeding values (EBVs) corresponding to 305-d yields, as follows:Mixed model equations were solved by the preconditioned conjugate gradient method using BLUPF90 . SolutioSM, SF, and SP contain covariables of five modified Legendre polynomials for DIM 5 to 305; Mi, Fi, and Pi are milk, fat, and protein yields of the first, second and third lactation, respectively. a α̂(1:5)i, α̂(6:10)i, and a α̂(11:15)i are five order of breeding value coefficient of animal ith for the first, second and third lactation as corresponding traits.where Genetic trends of the Thai dairy population were obtained by a regression of the yearly average animal (sires and cows), calculated average of breeding values of animals for that trait on that year. Obtain one number per year that was average of breeding value of animals on that year. With help of Excel 2010, software designed graphs showed genetic trend. Analyze regression model of SAS software was used to determine the signification of genetic trend. Genetic gain for any trait was estimated from averages of breeding values so that difference between averages of breeding values of population in each trait at the end and the beginning of period indicated genetic gain.The estimation of the daily AG, PE, and R variances in the first three lactations for milk, fat and protein yields are shown in The pattern of AG variances by DIM across lactation for milk, fat and protein yields are shown in The trends in the AG and PE variance estimates for all yields throughout lactation obtained in this study were comparable to the trends found by Miglior et al  and de RFor higher estimates of AG and PE variance in early lactation, we can discuss with postpartum condition. The transition period and the early lactation period are characterized by the mobilization of fat body reserves to cover the energy demands and include various metabolic and endocrine adaptations. Recent research has suggested considerable variation among high selected dairy cows for the release of energy fuels from stored adipose tissues . TammingThe average daily and 305-d yield heritability estimates for production yield traits in the first three lactations are shown in In general, the level and pattern of milk yield heritabilities obtained from RR models are sensitive to the model applied; Misztal et al  and otheHeritabilities of TD production yields along the lactation trajectory for selected DIM in the first three lactations are also illustrated in Estimated heritabilities for daily milk yield were moderate at the early stage, increased to the highest heritability at the middle stages after parturition, and then gradually decreased until the late stage of lactation, whereas estimated heritabilities for fat and protein yield were relatively high at the periphery of lactation. The heritability curves for all production yield traits were similar to those for Dutch dairy cattle reported by de Roos et al . HoweverThe AG and PE correlations of 305-d production yields in the first three lactations are shown in Results from our study were in accordance with those of Muir et al  and HammThe AG and PE correlations of production yields on different DIM within the first three lactations are shown in Estimates of AG correlations between production yields on the same DIM in the first three lactations are shown in Among various lactations, similar shapes of correlation curves at the same DIM were also reported by Strabel and Jamrozik  and HammIt should be noted that, in this study, there were no negative estimates between any of the selected DIM. Negative genetic correlations for selected DIM were reported in several studies ,13. The Changes in the average EBVs for different 305-day production yields of Thai dairy cattle in the first, second and third lactations, against birth year from 2001 to 2011, are presented in Genetic parameters of the production yield of the Thai dairy cattle population using a multiple lactation random regression test-day animal model with Legendre polynomials described a production curve with similar trends as those from major reports that used the identical model on Holstein populations. Yield traits in all lactations had moderately heritabilities and genetic correlations among production yields within lactations were higher than those across lactations, suggesting that these traits can be used as an important index trait to improve production yields in Thai dairy cattle through selection. Moreover, an improvement in high milk production will lead to the highest increase in fat and protein yields within lactations. Moreover, selection for high production yields in primiparous cows will lead to a further increase for multiparous cows. Selection of candidate animals should be made based on EBVs to improve the production yields under the conditions of Thailand. Clearly, the estimates for parameters that were produced in this study are, therefore, likely to be useful as a preliminary step to developing national genetic evaluations for the production traits of the Thai dairy cattle population. In addition, research on issues not addressed in this study, such as the heterogeneity of variances, will eventually be required for the implementation of an internationally accepted genetic evaluation system.


At the multivariate meta-regression, the effect of publication year was significant, as well as the effects of country, gender, and level. The impact of RT on isokinetic strength was not significant , whereas the impact of RT on throwing  was significant, as well as the effects of RT on isometric strength , on maximal strength , and on power . Conclusions: RT has a significant impact in handball players. Handball coaches could design conditioning protocols and programs based on our results. However, due to a number of shortcomings, including the high, statistically significant heterogeneity among studies and the evidence of publication bias, further high-quality investigations are needed.Purpose: Handball  is an intermittent and strenuous contact sport, the successful performance of which depends on frequent body contacts, and the ability to make repeated explosive muscular contractions required for jumping, acceleration, sprinting, turning, changing pace, and throwing a ball. Many studies have investigated the effect of resistance training (RT) in handball players, however with conflicting results. Therefore, our objective was to investigate the impact of RT on maximal strength (isometric and isokinetic strength), the power of both lower and upper limbs, and throwing velocity, in handball players. Methods: A comprehensive literature search yielded a pool of 18 studies, which were retained in the systematic review and meta-analysis. Results: A total of 275 handball players were included. The overall effect size (ES) of RT was 0.996 ([95%CI 0.827–1.165],  Team handball is a sport that requires physical efforts of high-intensity and of short duration with a strenuous contact and a particular ability to make and repeat explosive muscular contractions. During a handball match, more than 825 high intensity actions can be performed, requiring a high level of strength ,2,3,4. MThrowing is a fundamental skill in handball. Two basic factors influence the efficiency of shots: accuracy and throwing ball velocity. The faster the ball is thrown, the less time defenders and/or goal keeper have to save the shot. Coaches and scientists seem to have reached an agreement stating that the main determinants of throwing ball velocity are the timing of movement in consecutive body segments, the technique used, and the strength and power of both the upper and lower limbs ,2,3. Eacp < 0.001) in standing handball throwing velocity after 6 weeks of heavy upper limb RT. Furthermore, Hermassi et al. , p = 0.466) for isometric strength.Considering the control groups, the overall ES was 0.173  of standing throwing ball after 6 weeks of heavy upper limb RT programs. Moreover, van den Tillaar [By increasing workloads, introducing higher throwing weights (pyramid training), accompanied with a relatively small number of repetitions and less training time, is required to obtain an optimal response. Gorostiaga et al.  noted a  Tillaar , compariHowever, existing studies are mainly focused on concentric exercises ,29, evenDifferent studies  reportedA combination of muscle strength, handball techniques, and competitive skills training can significantly enhance maximal and specific explosive strength of the upper limb over an 8- to 10-week training program ,13,17,28In conclusion, throwing ball velocity can be increased positively after RT programs that incorporate, as a minimum, training two to three times per week for 5 weeks of general RT for the upper body or specific resistance protocols.p < 0.01), and in the upper extremity muscles (pec–deck) from 36.1 kg to 44.4 kg (p < 0.01). This increase occurred mainly during the first 2 weeks, whereas there was only a slight improvement during the following 4 weeks of training. The addition of a 6-week period of heavy training resulted in considerable gains in the maximal muscle strength of the leg extensors (13%) and the arm muscles (23%), with minor changes in the maximal isometric force of the leg flexor muscles (9%).Gorostiaga et al.  showed tA difference in strength gains between upper and lower extremity muscles can be explained by the difference in initial conditioning between knee extensor and upper body muscles, depending also on the pattern of quantity and/or intensity of daily physical use.Interestingly, studies investigating the effects of concurrent strength and endurance training found an interference in the optimal development of muscle strength . HandbalCompetitive performance in handball depends not only on muscle strength, but also on the ability to exert force at the speed required by this discipline. The longer contraction durations appear to be best suited for maximizing muscle strength . The traThese exercises, despite an initial explosive contraction, are performed at a slow velocity of movement and have the greatest effect on strength improvement at the slow velocity/high force segment of the force–velocity–power curve ,13. Howep < 0.01) and the leg extensors , although no gains can be anticipated from low resistance forms of activity such as normal handball practice. Furthermore, Hermassi et al. [In this context, Gorostiaga et al.  found thi et al.  and Cheli et al.  reportedi et al.  and Hermi et al. , possiblThe results of Hermassi et al. ,7,8 are p < 0.01) compared with T1. The greater number of weekly RT sessions could explain the higher increase in 1RM upper limb strength in this study.On the other hand, Gorostiaga et al.  studied Marques et al.  examinedpeak) determined by means of a cycling force–velocity test [p < 0.01) and upper  extremities [Few investigations in team handball examined the effects of RT using dynamic exercises on the peak muscle power  and relative (W/kg)  muscle power for the upper limbs, suggesting that muscle power gain could be attributable to an increase in the regional muscle volume of the upper limbs [Moreover, Hermassi et al.  investiger limbs .In fact, the average percentage increase in muscle power per unit of muscle volume (W/l) for the heavy training group (8.5%) tended to be higher than for controls (3.7%). Heavy RT may increase muscle bulk appreciably, potentially due to neuronal adaptation .p < 0.001) and in the relative power (W/kg)  of the upper limbs, but no changes when power was expressed per liter of upper limb muscle volume (W/l) [In fact, 12 weeks of heavy RT led to a considerable gain in muscle power (W)  . The aveAlthough the load prescription based upon the maximizing of mechanical power output appears an attractive strategy to enhance the muscle power of the limbs, performance may be critically dependent on the ability to exert force at speeds specific to a given athletic discipline. Longer contraction durations have been associated with heavier loads, so the prescription of such loads would seem best suited to maximizing strength.To the best of our knowledge, this is the first systematic review and meta-analysis that comprehensively and quantitatively assesses the impact of RT among handball players. However, despite its novelty, the present investigation suffers from a number of shortcomings which should be properly recognized and addressed. The main drawbacks are represented by the significant amount of heterogeneity and the evidence of publication bias that is present in many fields of the research domain.Our systematic review and meta-analysis showed that RT has a significant impact in handball players in terms of maximal strength (isokinetic and isometric strength), muscle power of both upper and lower limbs, and throwing velocity. The present review has important practical implications, in that handball coaches could design conditioning protocols based on our results. However, due to the above-mentioned shortcomings, including evidence of bias and high, statistically significant heterogeneity among studies, further high-quality investigations in the field are urgently needed.


The current study aimed to measure and decompose socioeconomic-related inequalities in DMFT  index among adults in Iran.The study data were extracted from the adult component of Prospective Epidemiological Research Studies in IrAN (PERSIAN) from 17 centers in 14 different provinces of Iran. DMFT score was used as a measure of dental caries among adults in Iran. The concentration curve and relative concentration index (RC) was used to quantify and decompose socioeconomic-related inequalities in DMFT.A total of 128,813 adults aged 35 and older were included in the study. The mean (Standard Deviation [SD]) score of D, M, F and DMFT of the adults was 3.3 (4.6), 12.6 (10.5), 2.1 (3.4) and 18.0 (9.5), respectively. The findings suggested that DMFT was mainly concentrated among the socioeconomically disadvantaged adults , − 0.066 to − 0.063). Socioeconomic status, being male, older age and being a widow or divorced were identified as the main factors contributing to the concentration of DMFT among the worse-off adults.It is recommended to focus on the dental caries status of socioeconomically disadvantaged groups in order to reduce socioeconomic-related inequality in oral health among Iranian adults. Reducing socioeconomic-related inequalities in dental caries should be accompanied by appropriate health promotion policies that focus actions on the fundamental socioeconomic causes of dental disease. Dental caries is one of the major public health concerns throughout the world. Poor oral health condition adversely affects the quality of life, oral health status and well-being of people . Dental Socioeconomic-related inequalities in various health outcomes constitute a main challenge for public health –6. AccorAlthough dental caries rates in the developed world are decreasing , 15–18, Iran, as a developing country, is located in the Eastern Mediterranean Region with an area of 1,648,000 km sq. Based on the 2016 census data, the total population of Iran was about 80 million people.In this cross-sectional study, we extracted and merged the required data from the adult component of the PERSIAN, which has been launched by the Ministry of Health and Medical Education (MoHME) to collect epidemiological data from 17 centers in 14 different provinces of Iran, since 2014, as follows: Kermanshah (KSH), Gilan (GI), Fars (FA), East Azerbaijan (EA), Mazandaran (MA), Sistan and Balouchestan (SB), Yazd (YA), Kerman (KE), Khouzestan (KH), Chaharmahal and Bakhtiari (CB), Hormozgan (HO), West Azerbaijan (WA), Ardabil (AR) and Razavi Khorasan (RK). While there is only one PERSIAN cohort center in 13 provinces, FA and RK have three  and two centers (Sabzevar and Mashhad), respectively. We obtained data from all the centers. The characteristics of cohort centers used in the study showed in The cohort questionnaire consisted of three parts of general, medical and nutrition with 482 questions. The first part included general questions on demographics, SES, lifestyle, occupational history, physical activity, sleep and circadian rhythm and mobile use. The second part consisted of questions related to medical issues . The third part asked questions regarding personal habits questions such as smoking, drinking alcohol and drug use. The cohort questionnaire was administrated by trained interviewers. Quality assurance (QA) and quality control (QC) measures were re-checked by the central and local QA/QC teams to ensure all procedures are performed in accordance with the PERSIAN Cohort protocol. The PERSIAN cohort is a cohort study that has different studying sites around Iran. Because of the coordination among these cohorts, the data collection tools and their definitions were comparable; therefore, we could compile their datasets with minimum conflicts. More details about the PERSIAN study can be found elsewhere , 24.The outcome variables was DMFT index as a measure of dental caries in the study . The DMFPrincipal component analysis (PCA) technique , 31 was We used both the concentration curve and relative concentration index (RC) to quantify and decompose socioeconomic-related inequalities in DMFT among Iranian adults (35 years and older) in the 14 provinces combined as well as in each province, separately. The RC is calculated based on the concentration curve, which graphs the cumulative percentage of participants ranked by SES (the constructed SES scores) on the x-axis and the cumulative percentage of a health variable of interest (DMFT score) on the y-axis. The RC is equivalent to twice the area between the line of perfect equality (45-degree line) and concentration curve. The values of the RC range from − 1 to + 1. If the concentration curve lies under (above) the line of perfect equality, the sign of the RC is positive (negative). The negative value of the RC indicated that DMFT score is more concentrated among rich vice versa. The value of zero suggested perfect equality .The following formula was used to calculate the RC:μ shows the mean of the outcome variable of interest  for the whole sample; yi presents the outcome variable (DMFT score) for individual i; and ri is the fractional rank in the SES distribution for individual i; (i based on the SES in the sample of n); and Where s the RC .y, to a set of k explanatory factors ,xk:The RC was decomposed to identify the main factors that contributed to the observed socioeconomic inequality in DMFT in the 14 Iranian provinces included in the study. Consider the following linear regression model that links DMFT score, Wagstaff et al.  showed tRCk is the RC for explanatory variables. The GCε indicates the generalized concentration index for the error term and it reflects socioeconomic-related inequality in DMFT that is not explained by explanatory variables included in the study All data analysis performed by Stata version 14.2  and p-value less than 0.05 was considered statistically significant.Where The descriptive statistics of all the variables used in the study are presented in Table The mean (SD) score of D, M, F and DMFT of the adults was 3.3 (4.6), 12.6 (10.5), 2.1 (3.4) and 18.0 (9.5), respectively. There is, however, variation among the provinces in the average score of DMFT score. As reported in Table Table The RC of D, M and F for the total sample was estimated to be − 0.1684 [95% CI: − 0.1726 to − 0.1642], − 0.1086 [95% CI: − 0.1111 to − 0.1061] and 0.4028 [95% CI: 0.3982 to 0.4075], respectively. The concentration curve of D, M, F and DMFT for the total sample is showed in the Fig. Figure Table The results of multivariable regression (the coefficients results) indicated that older age was associated with higher DMFT score. Compared to females, males had statistically significantly greater DMFT score. Also, the DMFT score among single was found to be lower than compared to other marital status groups. The mean of DMFT score was lower among people with better-off compared to socioeconomically disadvantaged individuals. Positive associations were found between unhealthy behaviors of smoking status and drinking alcohol and DMFT score. The results also suggested higher DMFT score among individuals residing in the provinces of GI, KSH, WA, RK, CB, KE, HO, KH, SB and MA than those living in FA province.RCk, were presented in the third column of Table RCk results indicated those who were male, married, smokers and drinker were relatively wealthier in the study population, whereas individuals who were divorced or widowed and older were relatively poor.The RC for each of explanatory variables, The term “contribution” shows how much the variation of each explanatory variable across SES groups can explain the observed association between SES and DMFT score. If the sign of contribution for a given explanatory factor is positive (negative), it suggests that the socioeconomic distribution of the factor and the association between this variable and DMFT score leads to a higher DMFT score among the worse-off (better-off). Based on the results reported in Table As reported in Table Dental caries is a major oral health problem in developed and developing countries. The current studies –13 also The average DMFT index was found to be 18.0 in 14 provinces in Iran with significant variation across provinces. We found statistically significant pro-rich inequality in DMFT score in all the provinces included in the study. Socioeconomic-related inequality in DMFT score was found to be large in provinces such as Ardabil, Yazd, Kerman, East Azarbaijan and Fars. A study by Moradi and collogues also indicated that the higher concentration of poor DMFT score among the poor in Kurdistan city, Iran . The meaBesides SES, our study also showed that being a female, older adults, married, smoking and drinking alcohol were associated with higher DMFT score among Iranian adults. Our study indicated that higher DMFT score among individuals residing in the cohorts of WA, AR, YA, KE, FA and EA compared to other provinces included in the study. A study by Piovesan et al.  also fouRCk for this variable Table RCk for these two variables in Table Beside SES, being male and older age and widow or divorced were the main factors contributing to the concentration of DMFT among the worse-off in Iran. The negative contribution of being male to socioeconomic inequality in DMFT is explained by the fact that men compared to women have lower DMFT score  in Iran; thus, the generalizability of our results to other provinces and other age groups is partially limited. Thirdly, the DMF score and its socioeconomic-related inequality can be influenced by other important factors such as ethnicity or nationality and living area . These factors, however, were excluded from the study due to the lack of data in the dataset used in the present study.This study revealed that dental caries, as measured by DMTF score, was concentrated among socioeconomically disadvantaged adults in Iran. We also observed significant variations in socioeconomic inequality in DMTF score among different provinces in Iran. As our study demonstrated SES, being a male, older age and being a widow or divorced as the main factors contributing to the concentration of DMFT among the worse-off in Iran, it is recommended to focus in the oral health status of these groups in order to reduce socioeconomic inequality in oral health among adults in Iran. For example, as the existing studies e.g., –50) show show50])


Tursiops truncatus), (2) describe the presence and characterization of heart murmurs detected in free-ranging and managed dolphins, and (3) characterize heart murmur etiology through echocardiography in free-ranging dolphins. For technique development, 65 dolphins cared for by the Navy Marine Mammal Program (Navy) were auscultated. The techniques were then applied to two free-ranging dolphin populations during capture-release health assessments: Sarasota Bay, Florida (SB), a reference population, and Barataria Bay, LA (BB), a well-studied population of dolphins impacted by the Deepwater Horizon oil spill. Systolic heart murmurs were detected at a frequent and similar prevalence in all dolphin populations examined , and characterized as fixed or dynamic. In all three populations, sternal cranial and left cranial were the most common locations for murmur point of maximal intensity (PMI). An in-water transthoracic echocardiogram technique was refined on a subset of Navy dolphins, and full echocardiographic exams were performed on 17 SB dolphins and 29 BB dolphins, of which, 40 had murmurs. Spectral Doppler was used to measure flow velocities across the outflow tracts, and almost all dolphins with audible murmurs had peak outflow velocities ≥1.6 m/s ; three dolphins also had medium mitral regurgitation which could be the source of their murmurs. The presence of audible murmurs in most of the free-ranging dolphins (88%) was attributed to high velocity blood flow as seen on echocardiography, similar to a phenomenon described in other athletic species. These innocent murmurs were generally characterized as Grade I-III systolic murmurs with PMI in the left or sternal cranial region. This study is the first to describe an efficient technique for in-water dolphin cardiac auscultation, and to present evidence that heart murmurs are common in bottlenose dolphins.Cardiac auscultation is an important, albeit underutilized tool in aquatic animal medicine due to the many challenges associated with in-water examinations. The aims of this prospective study were to (1) establish an efficient and repeatable in-water cardiac auscultation technique in bottlenose dolphins ( Deepwater Horizon (DWH) oil spill in 2010, cardiotoxic effects were documented in a variety of species, including fish (Tursiops truncatus) residing in Barataria Bay, LA (BB), numerous studies have described detrimental health impacts linked to DWH oil exposure, including pulmonary, reproductive, and endocrine disease , and echocardiography in living cetaceans, much of the knowledge regarding cetacean cardiology has been based on stranding, necropsy, and histologic findings –32, as wThere have been no peer-reviewed studies to date detailing cardiac auscultation and prevalence of heart murmurs in free-ranging or managed cetacean populations, nor studies describing the use of echocardiography in free-ranging dolphins. As such, the aims of the current study were to (1) establish an efficient and reproducible in-water cardiac auscultation technique in dolphins, (2) describe the presence and characterization of heart murmurs in both free-ranging and managed dolphins, and (3) better characterize heart murmur etiology through echocardiography in free-ranging dolphins.n = 15) also received echocardiography exams for technique refinement. Dolphins voluntarily participated in these procedures through positive operant conditioning. Navy dolphin participation was approved by the Navy Marine Mammal Program's Institutional Animal Care and Use Committee (NIWC Pacific IACUC # 128-2018) and from the U.S. Navy Bureau of Medicine and Surgery (NRD-1157). The Navy Marine Mammal Program is accredited by AAALAC International and adheres to the national standards of the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the Animal Welfare Act. One geriatric dolphin in this population has known cardiac disease (second degree atrioventricular block and mild aortic insufficiency).For technique development, 65 dolphins cared for by the U.S. Navy Marine Mammal Program in San Diego, CA participated in voluntary cardiac auscultation exams from March 2018 through August 2019, including 35 males and 30 females, ranging in age from 2 to 54 years, and 169 to 290 cm in total length, examined to be healthy during the period of the study. A subset of these dolphins  in June 2018, a non-oiled reference location, conducted under NMFS Scientific Research Permit No. 20455. All dolphins in this study were given complete veterinary examinations as previously described, and were assigned prognostic scores indicative of their overall health status , 51. NinCapture-released health assessments were performed in BB in July 2018, conducted under NMFS Scientific Research Permit No. 18786. Thirty-four dolphins were auscultated in BB, including 21 males and 13 females (6 confirmed pregnant on ultrasound), ranging from 195 to 267 cm in total length. Of the 34 BB dolphins auscultated, 29 also received echocardiograms. The methods of temporary capture and on-site release in both locations followed those previously described , 53.All auscultations were performed in seawater with the 3M™ Littman® Veterinary Master Classic II 32″ . The head of the stethoscope was covered in Parafilm® M laboratory film  and secured with electrical tape along the tubing for waterproofing. The duration of this waterproof seal was variable, and once a leak was identified, the stethoscope was traded out for another waterproofed stethoscope without a leak. The application of Parafilm® to the head did not affect the sound of the stethoscope.In all dolphins, cardiac auscultation began by manually palpating in between the pectoral flippers for the strongest heartbeat and placing the stethoscope on the body at that location. From there, six points of auscultation were standardized: sternal caudal, left caudal, left cranial, sternal cranial, right cranial, and right caudal , listeniThe timing of the murmur in relation to the heart sounds (systolic or diastolic) and in relation to the cardiac cycle (fixed or dynamic) was also determined. Dolphins normally have a pronounced respiratory sinus arrhythmia, with a “split” between the low and high heart rate phases. The lower heart rate phase occurs before the breath (bradycardic phase), and the heart rate increases transiently after the breath (tachycardic phase). The estimated low and high heart rates heard during auscultation were recorded for each animal. A fixed murmur was defined as one that did not change grade and was heard at the same level during the entire auscultation. A dynamic murmur was defined as one that changed grade within an auscultation period. Since dynamic murmurs fluctuated with the changing heart rates, they were given a grade in the bradycardic phase and in the tachycardic phase to reflect the changing intensity with heart rate.n = 65) were auscultated by authors BKL  and AH . Trainers held the dolphins in a voluntary right lateral presentation  in the water at the side of the dolphin's sea-pen enclosure. Veterinarians on the side of the enclosure then held the right pectoral flipper with one hand and auscultated the entire cardiac window with the other hand, allowing dolphins to comfortably roll to take breaths as needed  in addition to other authors . Veterinarians standing in the water beside the dolphin could reach under the water and gain access to the entire cardiac window with the dolphin upright . AuscultA 3M™ Littman® Electronic Stethoscope, Model 3200, was used to record heart sounds of Navy dolphins. Phonocardiograms were downloaded onto StethAssist™ Software  for analysis and identification of murmurs. Echocardiograms were performed on a subset of Navy dolphins to aid in technique refinement for free-ranging dolphin applications. Twenty-three in-water transthoracic echocardiograms were performed with 15 Navy dolphins. Dolphins were trained to assume dorsal, lateral, and 45-degree lateral recumbency with a trainer standing on a submerged platform. The examining cardiologist (AH or SH) stood on the platform beside the trainer and was able to scan the cardiac window in one or all of these three positions. Given that the aim of ultrasounding Navy dolphins was technique establishment for applications in the field, many of these exams were partial echocardiograms .In SB, 17 dolphins received in-water transthoracic echocardiograms (of 19 dolphins auscultated). In BB, 29 dolphins received in-water transthoracic echocardiograms (of 34 auscultated). Dolphins were gently restrained by experienced handlers in the water, keeping the dolphin mostly submerged with the blowhole clear of water. Echocardiograms were performed by cardiologists while standing beside the dolphin and reaching under to obtain access to the cardiac window. This was typically performed next to a boat, so that electronic equipment could stay dry and shaded on the boat. Dolphins were examined in dorsal presentation or in a 45-degree tilt to the right or left to allow for better visualization of the cardiac window. Most exams were completed in 5–7 min, and were performed opportunistically in the health assessment process, generally about 30 min from initial capture .All transthoracic echocardiograms were performed using the GE Vivid-iq ultrasound and GE 3S phased-array transducer cardiac probe (1.5–3.6 MHz) . The following standard echocardiogram images were examined: right parasternal long-axis, right parasternal short-axis, left parasternal apical, left parasternal cranial long-axis, left parasternal short-axis.Regurgitant jets were classified subjectively as none (absent), trace, small, medium, or large. Trace regurgitation had a barely detectable jet noted on color Doppler (a few color pixels). A small jet was one that was relatively narrow, that did not distend very far from the valve being interrogated, did not persist throughout the entirety of systole and/or appeared only mildly turbulent based on the mosaic color flow pattern. A medium jet was defined as one that appeared turbulent within at least ¼ of the associated chamber. A large jet was defined as one that appeared turbulent within more than ½ of the associated chamber. Classification criteria were defined by the investigators (SH and AH) based on standard evaluation of color flow jet origin, direction and size .To evaluate whether high outflow tract velocity causes murmurs in dolphins, the maximum blood flow velocity was determined across the left and right outflow tracts  and compared to 1.6 m/s, the speed above which high velocity flow typically causes an audible murmur in small animals due to enhanced blood turbulence at high velocity –61.Echocardiograms were analyzed using GE Vivid-iq ultrasound software, EchoPAC version 201. Two cardiologists (AH and SH) reviewed each study and made all qualitative and quantitative measurements. When measurements differed between cardiologists, they were discussed to reach a consensus.Murmur prevalence and type (fixed or dynamic) were compared between dolphin groups using two-sided Fisher's Exact Tests . In the cases where the prevalence of a finding in SB and BB was similar , the data were pooled into one “free-ranging” group for comparison with the Navy managed group. Other parameters were analyzed including murmur intensity grade, murmur PMI, maximum outflow tract velocity, and valvular regurgitation.p = 1, Fisher's Exact Test), nor between the managed and free-ranging groups (p = 0.54)  had heart murmurs. In SB, 17 of 19 dolphins (89%) had heart murmurs. In BB, 30 of the 34 dolphins (88%) had heart murmurs. All murmurs were systolic. There was no difference in murmur prevalence between SB and BB ( = 0.54) . In bothT-tests and adjusted p-values via the Benjamini and Hochberg method (p < 0.001).In Navy dolphins, the mean low heart rate during auscultation  was 49 beats/min (SD ± 12) and the mean high heart rate  was 78 beats/min (±12). In SB dolphins, the mean low heart rate was 73 beats/min (±11) and the mean high heart rate was 97 beats/min (±12). In BB dolphins, the mean low heart rate was 79 beats/min (±16) and the mean high heart rate was 103 beats/min (±15). Pairwise comparisons of the mean heart rates (both high and low) were performed across the three cohorts using g method . While tp = 0.22, Fisher's Exact Test). However, managed Navy dolphins had a higher prevalence of dynamic murmurs (85% dynamic and 15% fixed) when compared to free-ranging dolphins   .Fixed murmurs ranged from Grade I to IV. Dynamic murmur grades ranged from absent  to Grade II in the bradycardic phase and Grade I to III in the tachycardic phase. With increasing heart rates, dynamic murmurs became louder and grades increased in all cases . There wn = 7, BB n = 7), followed by left cranial , right cranial , and left caudal . In Navy dolphins, left cranial was the most common PMI (n = 26), followed closely by sternal cranial (n = 21), then right cranial (n = 5), left caudal (n = 4), sternal caudal (n = 3), and right caudal (n = 1). The sternal cranial and left cranial regions correspond to the basilar region of the heart, which was also noted to be the area of the strongest palpable heartbeat in most dolphins, and is in proximity to the aortic and pulmonic valves.In both SB and BB, sternal cranial was the most common PMI  was found to be the single best position for efficient auscultation in managed dolphins. With the dolphin upright in the water in a dorsal presentation, veterinarians were unable to reach to the sternum on larger dolphins from the site of the netted enclosure, and clinicians are typically not able to stand beside the dolphin in the water due to the depth of the enclosure. Therefore, a dorsal presentation in this setting was less efficient, as the dolphin and trainer had to reposition to listen to right and left sides of the sternum from the side of the enclosure in order to reach all of the auscultation points. In the right lateral presentation, all auscultation points could easily be reached and auscultated for several breath cycles. Auscultation findings in the right lateral position did not differ from those heard in left lateral positioning or dorsal presentation in the vast majority of dolphins. In two dolphins, right-sided auscultation was difficult in the right lateral present, so the heart was auscultated in both right and left lateral positions to hear both sides as closely as possible. In such instances, the outcome of auscultation  was the same regardless of position. Dorsal presentation was found to be the single best position for efficient auscultation in free-ranging dolphins, since veterinarians were standing in the water besides the animal and could easily reach the entire sternum without having to place the dolphins in lateral recumbency. Additionally, the free-ranging animals were not held in lateral recumbency as they are not trained for this behavior.Many of the dolphins exhibited high outflow tract velocities with no apparent stenosis. The maximum velocity ranges for the left and right outflow tracts were 0.98–2.7 m/s and 0.94–2.39 m/s, respectively. No evidence of stenosis (aortic or pulmonic) was seen in any of the dolphins. Of the free-ranging dolphins who had complete echocardiograms 46, 40 had heart murmurs. All but two dolphins with murmurs had outflow velocities ≥1.6 m/s . In three free-ranging dolphins, there was sufficient valvular regurgitation seen with color flow Doppler to be the potential source of the audible murmur. One dolphin in SB and two dolphins in BB were described as having medium mitral regurgitation based on color flow Doppler signal appearance. The murmurs in these three dolphins had differing classifications: the SB dolphin had a dynamic murmur, Grade II-III systolic, right cranial PMI, one BB dolphin had a fixed murmur, Grade III systolic, sternal cranial PMI, and the other BB dolphin had a fixed murmur, Grade III systolic, left caudal PMI. All three of these dolphins also had maximum outflow velocities ≥1.6 m/s. The vast majority of dolphins, however, had little to no structural or valvular changes on echocardiogram to account for the audible murmurs. Many dolphins were described as having trace or small regurgitant jets across all valves with no obvious evidence of hemodynamic remodeling to suggest significant cardiac disease. Five dolphins had no regurgitation or insufficiency and 39 had trace to small regurgitation or insufficiency at one or more valves.Therefore, 35/40 (88%) of the murmurs auscultated in free-ranging dolphins were likely due to high velocity blood flow across the outflow tracts. In three dolphins, the etiology of murmurs was potentially mitral regurgitation, though all three of these dolphins also had maximum outflow velocities ≥1.6 m/s.Cardiac auscultation is an important, albeit underutilized, diagnostic tool in aquatic animal medicine. It is an essential part of a complete veterinary physical exam in dolphins and a fundamental part of the cardiac exam. Early descriptions of dolphin cardiac auscultation described the dolphin heart as practically inaudible . DolphinA heart murmur is the audible turbulence caused by disruption of laminar blood flow within or in close proximity to the heart. Echocardiography is the gold standard for investigating the source of a murmur. Causes of heart murmurs can include pathological etiologies, such as primary valvular abnormalities , secondary valve dysfunction , or congenital defects . Many dolphins had trace or small valvular regurgitation; based on the prevalence of high outflow tract velocity and the consistently located PMIs, it is likely these trivial to mild regurgitations did not contribute to the audible murmurs. Trivial or mild regurgitation is often considered a variation of normal in human and veterinary echocardiography, and is also referred to as physiologic regurgitation , 61. In Given that the aim with Navy dolphins was technique development, many of these dolphins' echocardiograms were partial studies, and therefore the echocardiographic findings were not compared between free-ranging and Navy dolphins in the current study. Continued work to perform echocardiograms on additional Navy dolphins and further corroborate auscultation findings is ongoing. Thus far, the same appears to be true for Navy dolphins: most dolphins have low grade, dynamic, systolic murmurs with PMI left or sternal cranial and echocardiograms performed thus far have established that flow murmurs are also common in this population. In addition, it will be of interest in future work to compare Navy dolphins of varying activity levels, i.e., those who frequently deep dive in the open ocean, vs. those who occasionally swim in the open ocean, vs. those who do not. A flow murmur in these dolphins could be a sign of fitness, like in the highly trained sled dogs. Comparing murmurs between other populations of dolphins, beyond the three studied here and with varying activity levels, would also be of interest for future work.Using the standardized approach described herein, this study characterized murmurs previously not described in dolphins. It is unlikely that most of the murmurs are new and more plausible that these soft, dynamic murmurs were being missed previously due to brevity of auscultation, environmental factors, and/or lack of standardized technique. Underwater noise, patient movement, echolocating patients, water movement, and saltwater damage to stethoscopes all add difficulty to underwater auscultation. Many of the murmurs described were very subtle (Grades I and II) and could easily be missed due to loud ambient noise or an uncooperative patient. Listening to many dolphins repeatedly aided greatly in training the ears to diagnose these soft murmurs. It is also important to listen in the various locations for multiple breath cycles, as the murmur presence or intensity can change with the changing heart rate in a given location. Full cardiac auscultation as suggested may take several minutes per dolphin due to breath holding. Given these findings, this study highlights that performing auscultation for a longer duration and ideally with minimal ambient noise are the keys to proper cardiac auscultation assessment. Utilizing this technique for routine clinical assessment of Navy dolphins has yielded repeatable results in terms of murmur identification and characterization. While the majority of heart murmurs identified in dolphins in this study were determined to be innocent murmurs, it is important for clinicians in managed care settings to utilize repeated auscultation and monitor for changes over time. Murmurs with differing characteristics or murmurs changing over time may warrant further cardiac assessment, including echocardiography, and should be interpreted in light of other clinical findings.In summary, this report describes an efficient technique for in-water dolphin cardiac auscultation and presents the first evidence that heart murmurs are common in bottlenose dolphins. Heart murmurs were detected in most of the dolphins in this study, both managed and free-ranging, regardless of oil exposure. In the majority of dolphins with murmurs, structural abnormalities, such as valvular lesions or congenital defects were not identified, and the audible murmurs were attributed to high velocity blood flow seen on echocardiography, similar to the phenomenon described in other athletic species. These innocent murmurs generally were characterized as systolic, low grade (I-III), dynamic murmurs with PMI in the left or sternal cranial region; however, echocardiography remains the gold standard to definitively diagnose the etiology of a murmur.https://data.gulfresearchinitiative.org/data/R6.x809.000:0001.The datasets presented in this study can be found in online repositories. Data are publicly available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC) at The animal study was reviewed and approved by Naval Information Warfare Center Pacific's Institutional Animal Care and Use Committee, U.S. Navy Bureau of Medicine and Surgery, NOAA Animal Care and Use Committee, and Mote Marine Laboratory's Institutional Animal Care and Use Committee. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.BL, AH, FG, SH, LS, and CS contributed to the design and implementation of the research. BL, AH, FG, SH, RT, KC, VC, TR, AB, WM, CH, EZ, BB, FT, RW, LS, and CS implemented the free-ranging dolphin health assessments and data collection. BL, AH, SH, and VC implemented Navy dolphin exams in collaboration with EJ. RT, BL, AH, FG, SH, AB, WM, and KC performed data analysis. BL wrote the manuscript in consultation with AH, RT, FG, SH, and CS. All authors edited the manuscript.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. The handling editor declared a past co-authorship with AB and CH. The reviewer WV declared past-publications with KC to the handling editor.


Knee osteoarthritis (KOA) is a common degenerative joint disorder that affects 250 million people globally. KOA can lead to disability and is often associated with cardiovascular disease, poor quality of life, and mortality. The most common treatment for KOA is non-steroidal anti-inflammatory drug administration. However, the analgesic effect is limited and often accompanied by multiple side effects. Hence, many KOA patients opt for complementary and alternative medicine. Acupuncture is one of the most popular complementary treatments with great analgesic effect and minimal side effect. Electroacupuncture (EA) and laser acupuncture (LA) have been known to reduce pain in KOA patients. However, to date, no study has assessed the benefits of combining these two therapies.Fifty participants diagnosed with KOA, aged 50 years or older, and with consistent knee pain for more than 3 months were recruited and randomly assigned to the treatment group (EA plus LA) or control group (EA plus sham LA without laser output). All subjects in the treatment group will undergo a combined EA and LA treatment thrice a week for 4 weeks. The acupuncture will be performed on GB33, GB34, SP9, SP10, and ST36 sites. The treatment group will receive acupuncture with a transcutaneous electrical nerve stimulator at GB33, GB34, SP9, and SP10 sites and with LA at EX-LE5, ST35, and BL40 sites. The subjects in the control group will undergo the same treatment modality as the treatment group, except these subjects will not be exposed to laser output. Outcome measurements will include visual analog scale, Western Ontario McMaster Universities Osteoarthritis Index, Knee injury and osteoarthritis outcome, body composition analysis, knee range of motion, quadriceps muscle stiffness, one-leg standing with eyes open test, and the 30-s chair stand test before and after 4 weeks of intervention.This protocol aims to investigate the combined effect of EA and LA in KOA patients. KOA symptoms include knee stiffness, arthralgia, and limitation in range of motion, which can lead to a decrease in quality of life and even disability in severe cases.,4 A previous study showed that KOA also affects the structure of the knee joint. Synovial inflammation, abnormal bone structure, and bone marrow lesions are commonly observed in KOA patients, which could be the underlying cause of persistent knee pain. Among contributors to global disability, KOA ranks 11th, and some of its secondary diseases include obesity, cardiovascular disease, and leading to higher mortality.Knee osteoarthritis (KOA) is a common degenerative joint disorder that primarily affects elderly adults. In cases when such non-pharmacological interventions are inadequate, patients are often treated with topical non-steroidal anti-inflammatory drugs (NSAIDs). In moderate to severe cases, the treatment modalities include exercise, knee braces, walking aids, oral NSAIDs, and surgery. Oral NSAIDs are commonly used medicine to alleviate KOA-associated pain; however, long-term use of NSAIDs is often associated with cardiovascular diseases such as myocardial infarction, kidney injury, and gastrointestinal symptoms such as gastrointestinal bleeding.,15 Therefore, alternative treatment modalities to alleviate KOA symptoms are in high demand. Non-pharmacological KOA intervention includes exercise, heat and cooling packing, neuromuscular electrical stimulation, transcutaneous electrical nerve stimulation, low-level laser therapy (LLLT), massage, and acupuncture. The exercise programs emphasize on improving aerobic capacity, quadriceps muscle strength, and lower extremity performance.The modality of KOA treatment depends on the severity of its clinical symptoms. Currently, non-pharmacological interventions such as exercise and weight loss are the main treatment modalities for mild KOA. Electroacupuncture (EA), a combination of acupuncture and transcutaneous electrical nerve stimulation, has become a popular treatment for KOA and is associated with a low risk of adverse reaction.–21Acupuncture is one of the most popular treatment modalities to alleviate KOA symptoms. Transcutaneous electrical nerve stimulation and acupuncture have similar mechanisms to relieve pain without serious adverse effects.,23 Currently, LA is primarily used to address arthralgia or muscular pain.,25 LLLT has been reported to be a cost-effective analgesic therapy. However, the benefits of LA in alleviating KOA symptoms have remained elusive. In one previous clinical trial, LLLT has been suggested to alleviate KOA symptoms by increasing and decreasing serum beta-endorphin and substance P levels, respectively. Some studies analyzing the effect of LLLT in KOA patients produced ambiguous results. A meta-analysis on these studies conducted by Stausholm et al showed that LLLT can reduce pain and address disability in KOA patients. A randomized controlled trial conducted by Alghadir et al showed that LLLT was effective in reducing knee pain intensity at rest and movement, function, and ambulation. Therefore, LLLT seemed to be an effective modality for short-term pain relief and function improvement in patients with chronic knee OA.Laser acupuncture (LA), a form of LLLT, is painless, non-invasive, has no risk of infection, and can be combined with traditional acupuncture.To date, there are no studies on the effect of a combined application of EA and LA in KOA patients. We will conduct a two-arm parallel clinical trial. This study aims to investigate the effect of a combined application of EA and LA and explore the effects of LA in terms of knee pain, knee range of motion, knee stiffness, and quadriceps strength and function in KOA patients. We hypothesize that a combination of LA and EA will produce more beneficial results than the application of these therapies alone in addressing KOA symptoms.22.1The study was approved by the Human Ethics Committee of Chang Gung Medical Foundation Institutional Review Board, IRB No. 201901018A3C501. All participants will provide written informed consent before their enrollment in this study. Personal information will be collected, shared, and maintained in an independent closet to protect confidentiality before, during, and after the trial. The protocol has been registered at ClinicalTrials.gov (Identifier: NCT04188925).2.2This randomized controlled trial will be conducted at the Department of Chinese Medicine at the Kaohsiung Chang Gung Memorial Hospital from August 2019 to December 2020. The participants recruited from our outpatient departments will be randomly allocated to the treatment group  or the control group . All participants will receive 12 sessions of treatment over 4 weeks. The study design is depicted in Figure 2.3 and radiography . In this study, we recruited patients who were diagnosed with KOA, aged more than 50 years old and had knee pain for more than 3 months. Designated physicians evaluated the eligibility of all participants. Patients’ inclusion criteria for this study were based on the following factors: visual analog scale (VAS) > 3 points in daily activity, Kellgren–Lawrence Grading Scale grade is between 1 and 3 on knee X-ray, and informed consent obtained adequately. Patients’ exclusion criteria for this study were based on the following factors: history of knee arthroplasty, traumatic injury related joint deformity, body mass index more than 35, pacemaker insertion, photosensitivity, intra-articular injection or acupuncture 1 month before the enrolment, and any factors that may affect the evaluation of the outcome, such as psychiatric disorder and moderate to severe mental retardation.The diagnosis of KOA depends on the patient's history 2.4 We aimed to achieve an effect size of 0.569, a significance level (α) of 0.05, the desired power (1 − β) of 0.80, and correlation among repetition measures of 0.5, and as such, the estimated sample size for this study was 22. Anticipating a 20% dropout rate, a total of 50 participants were recruited.The necessary sample size was calculated from the result of a previous study on the effect of LLLT. In this study conducted by Al Rashoud et al, the mean difference (standard deviation) of the VAS at the baseline before the treatment were 6.4 (1.9) at the treatment group, and 5.9 (1.8) at the control group, and the mean difference (standard deviation) of the VAS after completing the treatment were 3.2 (1.7) at the treatment group, and 3.8 (2.3) at the control group.https://www.randomizer.org). Designated physicians enrolled participants who met the inclusion criteria and grouped the participants according to the result of randomizer by the website. The trial participants, outcome assessors, and data analyst will be blinded after assignment to interventions using labels A and B for the 2 groups.We used a randomized block design to distribute participants equally into two groups, A and B, with the help of a research randomizer , Knee injury and Osteoarthritis Outcome Score (KOOS), body composition analysis, knee range of motion test, quadriceps muscle stiffness, one-leg standing with eyes open test, and the 30-s chair stand test.2; power density, 5 W/cm2; and pulsed-wave; RJ-Laser, Reimers & Janssen GmbH, Waldkirch, Germany). The LA treatment will be applied to EX-LE5, ST35, and BL40  or sham LA and EA (the control group) over 4 weeks. LA will be performed using a gallium aluminum arsenide LaserPen  is verified by the Ministry of Health and Welfare in Taiwan. The acupuncture will be first performed on GB33, GB34, SP9, SP10, and ST36 sites Fig. . SubsequAfter treatments in each session, a designated physician will record the frequency of analgesic used, the VAS during rest and activity, the response to our therapy, and any adverse events. The patients can receive analgesics during the treatment period; however, they will be prohibited from receiving other acupuncture, rehabilitation program or knee surgery in any other clinic. If the patients do not follow these provisions, we will exclude these patients from our study protocol. On successful completion of 12 sessions, all participants will be subjected to the same exam and questionnaire as during the pretest.2.6We will evaluate the KOA patients with subjective tests and objective questionnaires at the beginning of the treatment and after 12 weeks of therapy. The data will be collected and entered into the patient's medical record in a computer, and all patient-related documents will be securely stored. Only the study-related investigators, including the physicians and the assistants will be allowed to access to the trial dataset. The primary outcomes will be the difference in the frequency of analgesic used and VAS scores during rest and activity between the treatment group and the control group. The secondary outcomes will be the difference between the control and treatment groups in terms of WOMAC, KOOS, body composition analysis, knee range of motion test, quadriceps muscle stiffness, one-leg standing with eyes open test, and the 30-s chair stand test after completing the 12 sessions of treatments. implying that an effective treatment will improve the VAS score by 1.8 points. WOMAC is a subscale for assessing the physical function of knee joints. There will be 17 questions with grading from 0 to 4 points for each question. Thus, the total score will be between the ranges of 0 to 68 points. Higher points correspond to more difficulties in performing a given activity. The MCID of WOMAC is 6 units. The KOOS is a self-administered scale and evaluates five outcomes, including pain, symptoms, activities of daily living, sport and recreation function, and knee-related quality of life. The scale is divided into five factions, including none, mild, moderate, severe, and extreme, which represent the severity of the deterioration of knee function and the impairment in daily living. A previous study showed that one-time LLLT had some effects on improving the knee function and muscle strength of quadriceps in KOA. We use Myoton-3 myometer and InBody 270 to measure muscle functions. In Myoton-3 myometer, the tip is placed on the midpoint of the patient's rectus femoris to measure muscle stiffness, representing the muscle ability to resist the changes of its shape, caused by external forces. InBody 270 can measure body composition, including the fraction of muscle and fat, and the muscle mass distribution of four limbs. This device will be used to evaluate the composition of thigh muscles and any possible muscle imbalance. One-leg standing with eyes open test will be used to assess balance and muscle strength of lower extremities. The patient will stand on one leg with their hands on hips and eyes opening for up to 120 s. We will record the time that the patient can stand on one leg under the instruction. Another functional assessment is the 30-s chair stand test, which can evaluate the muscle performance and the endurance of lower limbs. The patient will sit on the chair with bilateral feet stepping on the ground and hands crossed over the chest. Subsequently, the patient stands up and then sits down. Completing one standing up and then sitting down scores one, etc. We will calculate the number of times the patient can stand up and then sit down in 30 s. A designated study assistant will evaluate these tests.We will evaluate the frequency of analgesic used to treat knee pain after each intervention, including acetaminophen, a non-steroidal anti-inflammatory drug, or opioid drug. VAS will be used to evaluate the intensity of knee pain during activity and rest on a scale of 0 to 10, corresponding to the minimum and maximum discomfort intensity. The minimal clinical important difference (MCID) of VAS is 1.8 units,The reason for patients who are not completing the follow-up visit or dropping out of this study, such as adverse events, suboptimal response to therapy, failure to return for following up, and refusal to receive treatment will be recorded. The potential adverse events will also be recorded although only 6 J/acupoint will be delivered to each acupoint.2.7t test and chi-square tests will be used to assess and compare the baseline patient characteristics between the treatment and the control groups. Repeated measure analysis of variance and the generalized estimating equation will be used to evaluate changes in the VAS, WOMAC, KOOS, body composition, knee range of motion, rectus femoris stiffness, standing on one foot, and 30-s chair stand test between the treatment and the control groups. Statistical significance will be considered at a P-value of <.05. All statistical analyses will be performed with the SPSS for Windows, version 26 .All data will be presented in the form of mean ± standard deviation. The independent 2.8 However, since the designated acupuncture physicians are well trained in aseptic acupuncture procedures and proficient in human anatomy, the risk of serious complications is minimal. Additionally, the risk of fainting during the EA treatment will be extremely low because, in the study setting, the patient will be treated in a supine position.The IRB and Kaohsiung Chang Gund Memorial Hospital's statisticians will be a part of the data monitoring committee in this study. LA and EA are general practices. LA is a routine intervention and non-invasive treatment. The common side effects of EA are subcutaneous bleeding, pain, fainting during acupuncture treatment, while in very rare cases, serious complications such as pneumothorax or septicemia are possible.3,42 According to Taiwan's National Health Insurance Research Database, the frequency of KOA patients requiring total knee replacement has increased between 1996 and 2010. Also, the cost associated with total knee replacement has increased from $39.69 to $83.35 million. As such, it is important to identify cost-effective therapy for KOA; EA and LA are known cost-effective analgesic treatments.,43The prevalence of KOA in 50 years or older population is about 37% in Taiwan. This frequency is expected to increase, as Taiwan has a large number of elderly adults. EA cannot only alleviate pain and improve stiffness and disability, but also lead to a significant rise and fall in plasma beta-endorphin and plasma cortisol, respectively, after 10 sessions of treatment. Collectively, these effects could be the underlying analgesic mechanism of EA. This hypothesis is further validated by the fact that EA with etoricoxib was more effective than either placebo plus etoricoxib or etoricoxib alone in addressing KOA symptoms. Recently, a systematic review suggested that the therapeutic sessions of EA for at least 4 weeks significantly improved the VAS, WOMAC, and Lysholm knee score scale (LKSS).,46 A detailed literature review on the application of EA in KOA patients, including treatment frequency and assessment, is listed in Table Berman et al conducted a large randomized controlled trial study in 2004, which recruited 570 KOA patients and showed that EA had positive effects on improving knee function and relieving pain in KOA patients. The study concluded that a placebo effect was observed since the symptoms improved before and after interventions in both the treatment group and the control group. Another double-blind, randomized trial in 2004 with LLLT  at a frequency of 10 times in 2 weeks showed that there was no significant improvement in VAS and WOMAC parameters between the treatment group and control groups. A meta-analysis showed that LA had some positive effects on the WOMAC parameter in KOA patients; however, insufficient information on wavelength, energy density, and treatment duration made results’ interpretation difficult. In a recent meta-analysis, LLLT at a strength of 4 to 8 J and 785 to 860 nm wavelength with the mean duration of the treatment periods 3.53 weeks was found to reduce pain and disability in KOA patients after the end of therapy and during follow-ups 3 months. The multi-focal LLLT on quadriceps had shown not only analgesic effect but also improved function and muscular strength in KOA patients. The reasons that these studies exhibited ambiguous results may be the differences in laser dosage, laser power density, the treatment sites, and the treatment frequencies.A double-blind, randomized control trial in 1994 used a low power Ga–Al–As laser treatment in KOA patients at a frequency of 3 times per week for 3 weeks and showed that there was no difference in results between the treatment group and the control group. It is possible that EA treatments could lead to activation of the nervous system and inhibition of inflammatory and neuropathic pain by desensitizing peripheral nociceptors and reducing pro-inflammatory cytokines. The clinical effects of LA are likely to encompass anti-inflammatory, neural modulation, cell healing and regeneration effects. Both LA and EA have anti-inflammatory and neural modulation, which may decrease knee pain in KOA. Conversely, exercise therapy can reduce knee pain in patients with KOA and these exercise therapies are focused on improving aerobic capacity and quadriceps muscle strength. LA has an analgesic effect and can improve the function and muscle strength of quadriceps in KOA. Besides, LA and EA have many advantages, including low risks of complications, pain, cost, and high analgesic efficacy. Although some studies confirmed such beneficial effects of LA or EA in improving KOA symptoms, the combined effect of EA and LA on KOA remains elusive.Among many of the mechanisms of KOA, synovial inflammation (synovitis), abnormal bone structure, osteophytes, and bone marrow lesions can result in knee pain. The outcome evaluations will include analyses of subjective symptoms, quadriceps muscle functions, and body composition measurements. In conclusion, we hypothesize that a combination of LA and EA is expected to be more effective than these therapies alone in alleviating knee pain and stiffness and increasing muscle strength of lower limbs, which can further improve the balance and quality of life. Moreover, this combined therapy may also benefit in reducing the need for total knee replacement therapy and further decrease the cost coverage of national health insurance.In this study protocol, we will use LA at 6 J/acupoint delivered with an 810 nm laser in addition to EA treatment for 4 weeks. LA treatments are poised to heal damaged chondrocytes, which are more susceptible to pain stimulation.The authors would like to extend their appreciation to all the patients who participated in this study. We appreciate the assistance with the statistical analysis provided by the Biostatistics Center, Kaohsiung Chang Gung Memorial Hospital.SYW and NJC conceived, designed, and planed the study. SYW, CEK and WLH are recruiting the study participants and performing the interventions. WLH and YCH supervised the study. SYW, CHL, YT and CEK will interpret and analyze the data. CHL and SYW drafted the manuscript. CEK critically revised the manuscript for important intellectual content. All authors have full access to the manuscript and take responsibility for the study design. All authors have approved the manuscript and agree with submission.Chun-En Aurea Kuo: 0000-0002-1563-8651.Chun-En Aurea Kuo orcid: 0000-0002-1563-8651.