Grapevine grafting is an essential practice in viticulture and over the years, various bench grafting techniques have been developed to mechanize the nursery process and to increase the yield in number of viable cuttings. Bench grafting is a fundamental nursery practice that can potentially affect the quality of propagation material also in young decline associated to grapevine trunk diseases and has been recently reported to influence leaf symptoms development associated with diseases of Esca complex. The study aimed to investigate how three bench grafting methods  can influence these phenomena. Specifically, the different methods were compared for their effect on the anatomical development of the grafting point and the functionality of the xylem, also considering two factors: the grapevine cultivar  and the scion/rootstock diameter (thin and large). Observations by light microscopy on the anatomical evolution and measurements on the xylem morphology and hydraulic traits were correlated with the grafting methods and the investigated varieties. The anatomical observations revealed that the mechanical (Omega) and semi-mechanical (Full Cleft) grafting methods have a faster callusing response while the manual technique (Whip and Tongue) has a slower but greater vascularization of the differentiated callus. Significant differences between cultivars and/or grafting types were also detected in necrotic area on the grafted tissues. Statistical analysis of the grapevine vessels suggested differences in xylem parameters between cultivars, while grafting type had no significant effects. On the other hand, the grafting type significantly affected the intrinsic growth rate. The study confirms the potential incidence of lesions and dysfunctionalities correlated with the grafting method applied, which can potentially induce grafted vine declines in vineyards due to the necrotic area detected on the grafted tissues. Vitis vinifera scion and American Vitis spp. or interspecific hybrids of Vitis spp. rootstock, to form one composite organism  have been detected on grafting machines and during the grapevine propagation process . For thiThe occurrence of grapevine trunk pathogens in bench grafted vines has been reported by several authors . In commThe impact of the grafting practice in the grapevine decline associated to GTD was considered by some authors not only because favoring infections but also because of the influence of the physiology of the vine. Among such trunk diseases, Esca, a complex of diseases linked to fungal infections , 2022, hTherefore, the purpose of the present study was to investigate (i) Omega grafting as mechanical technique, (ii) Whip and Tongue grafting as manual technique, and (iii) FC grafting as semi-mechanical technique, comparing their impact on the anatomical development of the grafting point and comparing the functional integrity of the xylem following different grafting techniques by assessing hydraulic traits in the xylem vessels of the scion. To this aim also the incidence in necrotic tissue at the grafting point and the related potential risk of the young grapevine decline in vineyard were evaluated.Three methods of bench grafting were studied in the present investigation .® instrument  fixed on a grafting table  fixed on a grafting table  with similar caliber, variable between 7 and 10 mm. Two hundred and fifty grafts per cultivar were obtained through each bench grafting method.The scions of three diseases . Glera (V. vinifera cv. Glera (FEDIT 8 C.S.G. clone) were grafted on the rootstock V. berlandieri × V. riparia cv. Kober 5BB (FEDIT 101 C.S.G. clone), comparing two caliber ranges of propagation material: “thin grafted vines” obtained from scions and rootstock cuttings with caliber variable between 6 and 8 mm were compared to “thick grafted vines” obtained from scions and rootstock cuttings with caliber variable between 9 and 11 mm. Two hundred and fifty grafts per caliber range were obtained through each bench grafting method.The scions of the ®, Sipcam Italia Spa, Italy) for 30 min.In both trials, the propagation material was certified according to European Directive 2005/43/EC. After harvest from the mother fields, scion and rootstock canes were cut in scion buds (5 cm) and rootstock cuttings (30 cm) and during this operation no signs of tracheomycotic infections were detected on the base and on the top of rootstock cuttings and scion buds. Prior to grafting, propagation material was hydrated by soaking in water for 12 h, and disinfected by soaking the plant material in water containing 1 mL/L of thiophanate-methyl  and placed in callusing wood box covered with sawdust and agriperlite on the top of the box. Callusing took place for 15 days at 28°C and 85% relative humidity, during which a solution containing 2.5 mL/L of thiophanate-methyl , Italy) was applied as preventive treatment to control Botrytis cinerea. Callusing boxes were then left at environmental conditions for 10 days for the acclimatization of the callused grafted cuttings. Finally, callused plant material was removed from the boxes and selected by nursery operators in order to control: (i) the complete union between rootstock and scion and (ii) the callus uniformity. Grafted cuttings with incomplete junctions and non-uniform or hypertrophic callus were discarded. Selected grafted cuttings were planted in 6 L plastic pots with peat-rich standard soil . Potted grafted vines were grown under greenhouse conditions for 4 months (mid-June to mid-October): 2 L of water per pot were supplied weekly by drip irrigation and diseases controlled by spraying the foliage with a mix of 1.5 mL/L of copper hydroxide  and 40 mL/L of sulfur  every 15 days. Three additional treatments to control grapevine pests were performed during the growing stage by applying 2 mL/L of Acetamiprid , Italy).After being grafted according to each condition, grafted cuttings were waxed (Stähler Rebwachs Pron = 3) for each of the three types of grafts  per cultivar were selected randomly in Grafting trial 1. The same protocol was used for each of the six grafts  only per Glera cv. . Graft longitudinal sections (approximately 100 μm in thickness) were cut with a Sledge G.L.S.-1 microtome  and 4 months after T1 (T3). Three biological repetitions  equipped with a Nikon DS-Fi3 digital camera were used for qualitative and quantitative analysis. Lugol was used for starch staining .To evaluate the most successful grafting techniques to get an efficient connection between the two bionts in the Grafting trials 1 and 2, the following qualitative and quantitative parameters were used ; Table 1•Quality of the connection-level between scion (S) and rootstock (R) was evaluated on four categories of callogenesis. Category 4 shows a perfect connection between bionts, where callus covered the space between S and R and the graft line is practically unrecognizable. Category 3 shows a few structural imperfections in the graft border where the connection between the two bionts is almost complete. Category 2 is characterized by many discontinuities (wood or bark) in the border and in category 1 S and R are unattached and an intermediate space is clearly observed.•Anatomical part of bionts involved in grafting connection which means parenchymal tissue or cambium of S and R, respectively.•Internal callus (between bionts) differentiation grade.•External callus differentiation grade or the callus formed around the graft union (outside bionts).•Occurrence of necrosis areas in S or R, or in both of them.•Space between bionts.Necrosis areas were considered as degenerated tissues that appeared dark colored compared to the healthy tissue. These regions were measured by analyzing the callus growth at a quarter of the sample diameter, where the parenchymatic rays and cambium of the two bionts were observed. The necrosis areas were measured with Image J software 1.47v.® software and the following parameters measured: Xy area (μm2); lumen vessels area ; vessel density . The diameter of each vessel  was calculated as the diameter of a circle with an area equivalent to VA.In both trials, sections 2–3 cm long were cut from 1-year-old stems during dormancy. In trial 1, the 10th internode was selected for each combination of cultivars and grafting type while in trial 2, the 6th internode was sampled from Glera grafted cuttings. All the stem samples were placed in a 50:50 (v/v) mixture of ethanol and water and stored at 5°C. The stem sections were then fixed through ice on a Peltier plate, and transverse sections of 10–12 μm thickness cut using a rotary microtome . The secDh (μm) was calculated following The hydraulic weighted vessel diameter Dh incorporates the disproportionate contribution of large vessels to total flow and gives the average diameter needed for a given vessel density to result in the theoretical hydraulic conductivity for that stem Calculation of Kst) was calculated following The theoretical specific xylem hydraulic conductivity (–3 at 20°C); η is the viscosity of water (1.002 × 10–9 MPa s at 20°C), and Ai is the area of the image analyzed (m2).where ρ is the density of water  from day of the year (DOY) 182 to 281 (July 1st to October 8th). The relative growth rate (RGR) was calculated for internode number and plant height fitting biweekly measurements by Logistic functions at three parameters as follow:y corresponds to actual value of the measure, y0, initial value of measure (t0), r, intrinsic growth rate (1/time), k, carrying capacity , t, time. The parametrization of RGR were determined using growth rates R statistical packages software R 0.8.2 (where  R 0.8.2 .n = 5) while necrosis percentage was analyzed on three biological replicates (n = 3). For growth parametrization there were fifteen biological replicates per cultivar and grafting type (n = 15). Data were checked for normal distribution (D’Agostino–Pearson’s χ2 test) and homogeneity of variance (Levene tests). Percentages were transformed by arcsin function and normal distribution verified before analyses. In both trials, the effects of the cultivars and grafting type were assessed with two-way ANOVA. Post hoc analysis was conducted using Bonferroni test. Correlation was evaluated by performing Pearson’s test. All analyses were executed using dplyr, multcomp and car R statistical packages software R 4.0.2  than W&T grafting  . In all At T3, both Omega  and W&T At T1, W&T showed the best callogenesis which was mostly produced by both rootstock’s cambium and parenchymatic rays and only in part by scion cambium . In FC gAt T3, W&T still showed the highest callus production  , followeStatistical analysis on the necrosis percentage area indicated that significant differences were observed between cvs and/or grafting type . In partResults are presented according to each cultivar  grafted on the common rootstock Kober 5BB and the applied bench grafting method . No plant material was discarded by this operation; however, the callus feature was clearly different according to each bench grafting type, especially on cv. Glera: homogeneous callus in Omega grafted cuttings, homogeneous and hypertrophic callus in W&T grafted cuttings and less complete and hypertrophic callus in FC grafted cuttings.–2 vs. 120 and 121 mm–2 respectively). However, the significant interaction (P < 0.01) indicates that the Vd was affected by grafting type in relation to cultivars. Thus, Vd was significantly higher in the combination Glera-Omega than Cabernet Sauvignon-FC (137 ± 30.3 mm–2 and 85.2 ± 6.5 mm–2 respectively). Like Vd, the ratio vessel area: xylem area was significantly affected by cultivars. Glera had significant higher VAf than Teroldego and Cabernet Sauvignon (in average 22.4% vs. 19.7 and 18.6% respectively). On the contrary, hydraulic traits (Dh and Kst) were not affected by cultivar and grafting type. Dh ranged from 31.6 ± 3.1 and 25.7 ± 2.6 μm and Kst from 41.1 ± 12.7 and 26 ± 7.2 Kg Mpa–1 s–1 m–1. At the whole, a high vessel density determined a reduction of Dh (P = 0.02).Analysis of the grapevine vessels suggest differences in xylem traits between the three cultivars whilst grafting type did not have significant effects for all the considered traits . The culon of Dh . CorrelaR2 were higher than 0.98 for all fitted curves.Comparison of the parameters of the Logistic function revealed different patterns of stem growth dynamics relatively to cultivars for internode number and cultivar/grafting type for plant height measurements . R2 wereK (P < 0.0001). Cabernet Sauvignon showed a higher K and r coefficients than Teroldego and Glera (30.2 vs. 25.8 and 22.7 number of internodes respectively). However, the significant interaction (P < 0.01) for K coefficient indicates that the final number of internodes per plants was affected by grafting type within each cultivar. The Omega and FC grafting induced the higher number of internodes in Cabernet Sauvignon (in average more than 30 internodes each) and the lower in Glera (in average 23 internodes). Teroldego and Cabernet Sauvignon had significantly higher intrinsic growth rate than Glera .As shown in P < 0.0001) and grafting type (P = 0.0001). Teroldego and Cabernet Sauvignon showed the higher K than Glera  whilst FC determined significant high K coefficients than Omega (102.6 vs. 81.2 cm respectively). The intrinsic growth rate, r, was significantly affected by grafting type (P = 0.001). Omega grafting determined a higher r for plant height than FC and W&T.As shown in Vitis vinifera L. cv. Glera (FEDIT 8 C.S.G. clone) was grafted on the rootstock V. berlandieri × V. riparia cv. Kober 5BB (FEDIT 101 C.S.G. clone).At T1, FC-thin showed the highest connection between bionts  in compaAt T3, Omega thin showed the best connection between bionts  where thRegarding necrosis area % in Glera, Omega thin grafting showed the lowest value (9.7 ± 1.3) compared to the W&T and FC of the same caliber. Considering the thick caliber, the necrosis areas were not significantly different with value comparable to those observed in both W&T and FC thin .Results are presented according to the applied bench grafting method  and both caliber ranges (thin and thick) of Glera grafted on Kober 5BB. Few grafted cuttings were discarded after callusing: two Omega grafted cuttings for both caliber ranges did not present complete callus; one thin W&T grafted cutting with uncomplete and hypertrophic callus; as well as four thin FC grafted cuttings. In general, thick grafted cuttings showed a more homogeneous callus although more hypertrophic than thin grafted cuttings. The callus of FC grafted cuttings was less complete and hypertrophic in both caliber ranges than in the other conditions.Kst calculated on the 6th internode was similar between grafting types showing a negligible effect of the size of scion on the xylem morphology and hydraulic efficiency at the end of growing season.The effects of the caliber ranges (thin and thick) on hydraulic traits of Glera grafted cuttings were reported in The experiments here reported aimed to investigate on possible changes induced on anatomical and hydraulic traits of just grafted vines by applying three different grafting methods on three different cvs. Field observations by It is well known that after intimate contact between bionts, a new parenchymatous tissue occurs as a common response to wounding . One of Considering the impact of the three grafting techniques on the successful connection between bionts at T3, Omega grafting still showed high distance (about 2 mm) between bionts and the connection was guaranteed by a differentiated callus filling this space, confirming the essential role of this tissue during the grafting union formation . This waPrunus domestica as well as heterografts of Prunus cerasus cvs where cell differentiation and xylogenesis led to the formation of vascular connections  were significantly influenced by cultivar (P < 0.0001). However, the significant interaction cultivars x grafting type (P = 0.03) demonstrated that the grafting type had different effect within each cultivar. The post hoc analyses showed that combination of Glera-Omega and Cabernet Sauvignon-Full Cleft determined significant changes in VD . The effect of cultivars on the xylem morphology was already reported in grapevine . With the same xylem area, the high vessel surface could determine a reduction of wood basal density as already reported in other woody species  should have wider Dh than cvs with high VD (Teroldego and Glera). It is well known that larger vessels assure higher hydraulic efficiency (lower resistance to water flow) than smaller ones (V. vinifera cvs (P = 0.02) considering the three cvs pooled.Hydraulic transport efficiency of the xylem is one of the most important traits determining the growth performance of a species as well as its capacity to respond to environmental constrains. The results of trial 1 showed that the vessel density (VD) expressed as number of vessels: xylem area ratio (excluding phloem and ray parenchyma) as well as the total vessel area: xylem area ratio . This discrepancy in the results can be explained by the sampling of internode position along the stem. In fact, in this trial we sampled the 6th internode, while in the first trial we sampled the 10th internode in the apical part of the stem.Our results showed that grafting type did not induce significant changes in vessel density and hydraulic traits. In this experiment, Glera plants had lower VD than what recorded in the first trial  was affected by cultivars type for internode numbers and by grafting type for plant height. Intrinsic growth rate is an important parameter for many ecological applications, such as population risk assessment and harvest management  as consequences of their high r. The significant effect of grafting type on r, supported the hypothesis that grafting type was able to modulate stem growth in grapevine. In particular, Omega type grafting determined a significant higher r than W&T and FC for the plant height. Contrary to our expectations, the high r did not correspond to final taller plants. This discrepancy could be explained by the presence of time lag in growth in Omega grafted plants that we were not able to detect for the longer timing range between measurements. These results emphasized the possible effect of the grafting type on the plant phenology.The estimation of the parameters of the Logistic curves related to the number of internodes and plant height allowed to compare the growth trend for all the cultivars × grafting type combinations during the trial 1. Our results showed that the intrinsic growth rate  and semi-mechanical (Full Cleft) grafting methods have a faster callusing response while the manual technique (Whip and Tongue) has a slower but greater vascularisation of the differentiated callus, especially in the first growing time, immediately after callusing, which was found to be the most critical stage.The grafting methods and the grapevine cultivars studied were confirmed to impact the scion hydraulic traits and growth during the first vegetative season, independently from the anatomical differences initially detected after callusing. Considering that a stem expressing extensive hydraulic failure could be more prone to express GTDs related symptoms in vineyard or, in the worst cases, to die , the detIn this view, the results suggest to extend the study on the same propagation material planted in vineyard, throw mid-term observations in the mature and productive vines, in order to verify the relationship between the anatomical characters and the hydraulic traits with the young grapevine decline and GTDs incidence.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.EB and SF: conceptualization, methodology, formal analysis, investigation, data curation, and writing – review and editing. AG and SS: conceptualization, resources, methodology, formal analysis, investigation, data curation, and writing – review and editing. CT: methodology, formal analysis, investigation, and data curation. RP: investigation and data curation. AP: data curation, resources, and writing – review and editing. SD: conceptualization, methodology, and supervision. LM: conceptualization, methodology, resources, and writing – original draft, review and editing, supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.


Further, clinico-pathology, treatment and management measures in HNPV envenomed dogs are under recognized. Prospective investigations were performed to assess the clinico-pathology and management options for HNPV envenomed dogs brought to the University of Peradeniya’s Veterinary Teaching Hospital from January, 2012 to March 2018. We recorded the local and systemic manifestations, hematological and urinary abnormalities of 78 dogs in which HNPV bite had been witnessed by the owner. Mild swelling, extensive swelling, hemorrhagic blistering and hemorrhagic bullae at the site of bite were observed in 59%, 31%, 6% and 4% of the dogs, respectively. Some dogs were subjected to surgical excision of necrotized tissue including limb amputation. We observed the following systemic clinical effects in envenomed dogs: neurotoxicity (13%), acute kidney injury (AKI) (14%) and coagulopathy (16%). All dogs showed leukocytosis with mean white blood cell count of 25.25 × 103/µL. Mild anemia and thrombocytopenia were detected in 29% of the dogs. There was a significant correlation between extent of local tissue injuries with length of hospitalization (LH). The mean time of coagulopathy observed was 21.3 h (IQR: 8–48 h). In coagulopathic dogs, there was a strong correlation between LH and extent of local tissue injury ; LH and whole blood clotting time(CT) ; PT and aPTT . LH was significantly correlated with the development of AKI (p = 0.0013). Lack of specific antivenom (AVS) for HNPV envenoming provided an opportunity to study the remaining treatment options. Therefore, the study allowed the identification of local and systemic effects, hematological abnormalities, possible supportive treatments and drawbacks of management measures for envenomed dogs.Human envenoming from the bite of the abundant hump-nosed pit viper (Hypnale  Hypnale are the smallest of the known pit vipers  and inhabit Sri Lanka and the Western Ghats mountains of the Indian peninsula2. Historically, there has been controversy and disagreement about which species should be included in the genus Hypnale. According to the taxonomy established by Fitzinger in 1843, the Hump-nosed pit viper is represented in the genus Hypnale. Consequently, three species that belong to Genus Hypnale have been identified namely, Hypnale hypnale, Hypnale nepa and Hypnale zara. H. hypnale is widely distributed across the island, while H. nepa can be found in the Central Highlands and H.zara in the South Western wet zone low lands3. Further, H. nepa and H.zara are endemic to Sri Lanka3. Forest and anthropogenic environments are the commonest habitats of H. hypnale and H. nepa, but H. zara is restricted to lowland rainforests3.Hump nosed pit-vipers (HNPV) of the genus 7. However, HNPV envenoming has a low comparative incidence in dogs8. H. hypnale is one of the first snakes for which the pathophysiology of envenoming was scientifically studied. Davy in 1821 reported local swelling, deep ulceration and bloody stool resulting from envenoming in dogs, chickens and frogs9.Although human fatalities from HNPV bite are infrequent they occur across a widely dispersed area in Sri Lanka and South-Western India, a high incidence being reportedHypnale spp. venoms demonstrated severe cytotoxicity by inhibiting cell proliferation in invitro in chick biventer cervicis preparations and confirmed lesser neurotoxicity, myotoxicity and procoagulant activity10. Further, in this study, Hypnale spp venoms exhibited low IC50 values compared to cytotoxic Naja mossambica and Australasian death adders indicating that the venom contains cytotoxins of greater potentcy compared with other snake venoms. Proximal tubular cell injury and acute tubular necrosis with intact basement membrane have been observed in mouse models indicating possible direct nephrotoxicity following HNPV envenoming11.13 but 10% of victims suffer kidney injury14 and death following acute kidney injury has been documented in man16. According to the recent studies conducted17Hypnale spp. is considered as a highly venomous snake which needs antivenom as death have been reported in man following HNPVs bite. Unlike the bites from other species of venomous snakes, the occurrences of systemic effects are rare and unpredictable with HNPV bites2. The rarity and unpredictability of the sporadic occurrence of these potentially fatal systemic effects of envenoming by HNPV have made it an important species for investigation18. Moreover, the presence of nephrotoxic properties has been reported from H. hypnale, and from H. zara, but the nephrotoxic effect from H. nepa bite is as yet unknown15. Cardiac involvement has been recorded with transient ECG changes but evidence of myocardial trauma has not been proven due to normal levels of cardiac troponin in HNPV envenoming in humans19. However, HNPV venom has been shown to cause lung pathology10. In Sri Lanka, the Indian polyvalent antivenom (AVS) currently in use to treat venomous snake bites in man does not include HNPV antivenom. In addition, a venom quantification study was able to demonstrated venom antigen in circulation up to 24 h post bite in human due to scarcity of specific antivenom for Hypnale spp. envenoming20.Predominately HNPV bites in humans cause local effects such as pain and swelling, local hemorrhagic blistering, regional lymphadenopathy and rarely severe local necrosis and gangrenenaja and Daboia russelii envenoming in dogs have been established recently22. However, detailed investigations beyond several fundamental studies related to HNPV bite envenoming are scarce. The benefit of early surgical intervention to minimize the extent of local tissue injury due to H. hypnale envenoming in dogs has been suggested in one study23. However, the consequences of HNPV in dogs has not been studied in depth, and venom concentration has not been measured. Hence, there is currently a paucity of clinical profiles, treatment and management plans for dogs in Sri Lanka. This study utilised HNPV envenomed dogs to investigate the role of HNPV venom in the observed clinical profile and biochemical changes in body fluids. Crucially, these studies provide evidence of a hitherto unidentified action of H. hypnale venom.Treatment protocols using AVS for Naja Hypnale hypnale) envenoming were studied prospectively from January 1, 2012, to March 31, 2018. The study was conducted in accordance with relevant guidelines and regulations. Three methods were used to recognize offending snakes viz visual identification of snake specimens brought by dog owners;23; identification through photographs of the culprit snakes taken on mobile phones by owners and, identification by owners through photographs and preserved specimens of the culprit snakes. All the clinical examination procedures were performed in the intensive care unit of the VTH. The study was conducted after the informed consent was given by the dog owners. In order to find out the modality of HNPV envenoming detailed clinical examinations were performed on all patients. All the body systems were examined and qualitative and quantitative measurements of the clinical signs and symptoms were recorded. In order to classify the extent of tissue injury/s at the site/s of bite the grading system explained in the studies conducted by Grassi et al., 2016 and Adhikari et al., 2020 were used in this study24. Accordingly, severity of local tissue reactions at the site of bite of the patients were graded.Dogs that presented to the emergency service of the Veterinary Teaching Hospital (VTH) of the University of Peradeniya following hump-nosed pit viper (n = 78) before initiation of treatment. 2.5 mL of blood was placed in a plastic tube containing Ethylenediamine tetraacetic acid (EDTA) 1.5–2 mg/mL as the anticoagulant to perform full blood count (FBC), alanine aminotransferase (ALT), total protein (TP), aspartate aminotransferase (AST), Albumin, fibrinogen Blood Urea Nitrogen (BUN) and creatinine values. The same tests were repeated 24 h after the initiation of supportive treatments. A volume of 0.9 mL of blood was collected into a tube containing 3.2% tri-sodium citrate at a ratio of 9:1 to perform Partial Thromboplastin Time (PT) and Activated Partial Thromboplastin Time (aPTT). The sample was then centrifuged at 2000 g for 10 min, and plasma was separated. The semi-automated biochemistry analyser  was used to perform PT and aPTT tests. About 1 mL of blood was placed in a Khan tube to measure the clotting time (CT). Moreover, the exact test is termed as whole blood clotting time (WBCT20) in human medicine4.On admission, blood and urine samples were collected for hematological, biochemical tests and urinalysis respectively, which were repeated until they normalized. Blood samples were collected by venipuncture  from all the patients , chlorpheniramine maleate  0.4 mg/kg, IV, q.i.d.), cloxacillin sodium , metronidazole , and fluids  were administered intravenously for the initial 24 h of hospitalization. The treatments were continued until the patients recovered. The currently available AVS does not include antibodies for the venom of HNPV. Therefore, none of the patients were treated with AVS.Wounds with necrotic tissue, and heamorrhagic blisters, when observed, were subjected to treatment with the aim of reducing the amount of necrotic tissue and further aggravation of necrosis. The necrosed tissue was surgically excised at the site of bite under general anesthesia. Supportive treatment was initiated before such surgical intervention and intravenous isotonic saline was continued in order to stabilize the patients for general anesthesia. Diazepam  was given as anaesthetic pre medication. Propofol; , an ultra-short-acting compound was given to induce general anesthesia and maintained throughout the procedure using propofol at the rate of 0.1 mg/kg/min. The surgical site was prepared using isopropyl alcohol, chlorhexidine and povidone iodine, in a standard manner. Surgical excision of the blistered area with necrotized tissue at the edges of the wound area was performed. Myotomy was performed and devitalized tissue were removed as required. Muscles and subcutaneous tissues were sutured using 2/0 chromic catgut and skin was sutured using suture nylon, applying the simple interrupted suture pattern. The same antibiotic combinations were continued until completion of wound healing. On examination, a hemorrhagic bullae was observed in the left medial entire tibia-fibula region and the bullae has ruptured exposing the medial aspect of the entire shaft of both tibia and fibula  were observed isotonic saline (0.9%) was administered by intravenous infusion to ensure adequate urine output. Patients were observed for the production of urine and if anuric or oliguric, furosemide  Ltd, India; 2 mg/kg IV, 4 mg/kg or 6 mg/kg IV) was administered and observed for one hour to determine the production of urine. If there was no diuretic effect, then furosemide was repeated at the dose rate of 4 mg/kg IV.Data management and descriptive statistics were performed using MINITAB 16 software and Microsoft Excel (Excel 2010). The Spearman’s rank correlation was used to find relationships between variables . Comparisons between groups  for time lapse and the duration of hospital stay as dependant variables were done using Wilcoxon Rank Sum test. Comparisons among the groups  on the time lapse and the duration of hospital stay as dependant variables were done using Kruskal–Wallis test. All the interpretations  were made at the α = 0.05, unless stated.Ethical clearance was obtained from the Ethical Review Committee of the Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka , which is on par with the international standards of ethics on animal experimentation. All experimental procedures and animal care were approved by the Faculty Ethics Committee, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka.We certify this manuscript has not been published elsewhere and is not submitted to another Journal. All authors have approved the manuscript and agreed with submission to Journal of Veterinary Research.n = 61 (78%) were reported during 0500–0700 h and 1700–2000 h.There were 78 patients reported during the study and 68 of them were males (87%), and 66 (85%) were below the age of one year. The median age was 6.5 months and IQR was 4–10.25 months. The majority of bites n = 68 (87%). Fang marks were observed in medial aspect of hind limbs in n = 11 (14%) of dogs. Mild swelling, extensive swelling, hemorrhagic blistering and hemorrhagic bullae were observed in these patients. If the affected anatomical site was swollen to an additional half of its normal size, it was recorded as extensive swelling. The length of hospitalization was increased with the progression of tissue injuries  and neck regions Dogs with hemorrhagic blistering at the site of bite  was subjected to surgical excision of necrotic tissue by fasciotomy and myotomy under general anesthesia to minimize further aggravation from necrosis. In n = 1 (0.78%) patient with a ruptured heamorrhagic bullae and subsequent exposure of the shaft of the medial side of the left tibia fibula, mid femoral amputation was required.Systemic manifestations of neurotoxicity, hypersalivation, hyperesthesia and dilated pupils, were observed in 10 dogs (13%). The demographic and coagulation parameters of dogs with HNPV bite envenoming are presented in Table Coagulopathy was suggested in 16 dogs 21%) with evidence of prolonged PT, aPTT, CT and hypofibrinogenemia; although bleeding from the bite site or injection site was not observed. The mean time required for development of coagulopathy was 21.3 h (IQR: 8–48 h). Moreover, the mean time lapse from bite, LH, PT, aPTT and CT were higher in dogs with coagulopathy than in the non-coagulopathic dog group. The mean LH of dogs with coagulopathy was greater (11.3 days) than that of non-coagulopathic dogs (6.6 days). The lowest fibrinogen concentration was observed in the coagulopathic dog group ; length of hospitalization and CT ; PT and aPTT . In non-coagulopathic dogs, correlation between fibrinogen concentration and aPTT was significant  while other variables were not correlated.For coagulopathic dogs, correlations between the time lapse, LH, extent of injury, PT, aPTT, CT and fibrinogen were not significant Table , except p = 0.0013). However, dogs with and without kidney injuries had similar time lapse for admission (p = 0.374).Nephrotoxicity was observed in 11 dogs 14%). AKI was indicated by oliguria with impaired kidney functions within 24 h after admission . However, the length of hospitalization of dogs with local manifestations was shorter (5.3 days) than that of the dogs with systemic manifestations (11.2 days) (p < 0.0001). All the patients recovered only with supportive treatments without administration of AVS. However, due to the necrotic effects of HNPV venom 10% of patients suffered permanent disabilities.Except leukocytosis, 48 dogs showed only local clinical manifestations while 30 dogs showed systemic manifestations and/or deviated laboratory test results. There was no significant difference in the time lapse for hospitalization between the dogs with either local and systemic manifestations olysis35, and also the venom showed direct thrombin-like enzyme activity on fibrinogen36. P-I protease contributes to local tissue symptoms in H. hypnale envenoming in 90% of cases of man32. The clinical manifestations are inflammation (swelling and pain), wound hemorrhage, blistering, and irreversible tissue destruction i.e. necrosis, and involve disruption of capillary vessel integrity, matrix degradation, dermal–epidermal junction separating and secretion of proinflammatory mediators ie leading to tissue ischemia, disrupted tissue repair, and a varied degree of tissue damages 32. Mild swelling and extensive swelling observed in this study are consistent with the observation of envenoming in dogs due to N. naja in Sri Lanka21. Further similarity of varying degrees of swelling, haemorrhagic blisters, local tissue necrosis and regional lymphadenopathy are reported in different studies in human victims of HNPV bite envenoming15. Mild and extensive swelling at the site of the bite resolved spontaneously without any adverse outcome as observed in previous studies in both man and animals37. However, to address snakebite-induced local tissue damage and implement wound care, surgical management, including limb amputation on rare situations, had to be employed for HNPV envenomed dogs, as has been necessary for N.naja envenomed dogs21. As well as palliative treatment measures, wound care, surgical intervention, skin grafting and occasional limb amputation, are practiced for HNPV envenomed human patients1. There is thus, some evidence that early surgical intervention for such dogs is beneficial to minimize tissue necrosis, cost of treatment and to improve quality of life for dogs. However, in addition to the risk of mortality, a venomous bite by a member of Viperidae or Elapidae families also causes a severe local tissue trauma which frequently leads to amputation of the bitten limb to save the life of the victim38. Therefore, surviving victims suffer a substantially-reduced quality of life due to the multiple effects of permanent physical and psychological disabilities. Hence, exploring the potential utility of small molecules as community-based therapeutics to prevent local tissue damage following snakebite will help to prevent and minimize the extent of local tissue damage.Snake venom metalloproteases are Zinc-dependent metalloproteases and, are classified into four different protease sub classes: P-I, P-II, P-III and P-IV, almost as abundant as phospholipase A2 in 39. However, neurotoxicity have not been reported in humans patients4. In humans, systemic effects: nephrotoxicity, coagulopathy, thrombocytopenia and spontaneous haemorrhage, as well as less common effects  are less common39. However, despite being rare the unpredictability of sporadic and potentially fatal systemic effects of HNPV envenoming confirms the significance of this species31. Similar to the current study, systemic clinical signs of coagulopathy and AKI have been reported, although not frequently, in HNPV bite envenoming in humans40.The development of systemic manifestations was fewer compared to local clinical signs in patients in this study. Severe systemic envenoming of coagulopathy, AKI and neurotoxicity, were present in only approximately 20% of victims. Interestingly, neurological manifestations were the least common systemic manifestions reported in this study. Moreover, focal neuronal degeneration in brain have been reported in mice modelsp < 0 0.0001); length of hospitalization and CT ; PT and aPTT  were observed in dogs with coagulopathy. Therefore, care must be taken when managing patients with coagulopathy. The effects of HNPV venom on coagulation factors and renal tissues in this study contrasts with the pronounced effects of elevated PT, aPTT, CT, BUN, creatinine and decreased concertation of fibrinogen. Therefore, it is crucial that all the HNPV bitten dogs are observed and have investigations done to assess the presence of coagulopathy or AKI. Further, CT is a very simple, cost effective bed site test which can be used to detect VICC even in resource poor clinical settings. Accordingly, it is important to note that the more subtle effects of HNPV venom on the coagulation pathway and renal tissues may yet be found by more detailed biochemical analysis since the mechanism of AKI in HNPV envenoming remains unidentified2.Positive correlations between LH and extent of local tissue injury (rs = 0.78, N. naja bite envenoming ad D.russelii bite envenoming in dogs and humans.41. However, the cause of leukocytosis following such snakebite envenoming is yet to be explored22 and elucidation of this parameter would have the potential to open avenues to estimate the severity of envenoming22.Currently available AVS in Sri Lanka is imported from India and uses venom from the Indian species of Naja. naja, Bangarus caeruleus, D. russelii and Echis carinatus. HNPV, which is common in Sri Lanka and South East Asia is not included in the immunization mixtures used in the production of this AVS42. Scarcity of AVS has resulted in quantitative measurement of venom concentration within 24 h of snakebite20. Therefore, development of antivenom against the deadliest venomous snakes in Sri Lanka must include venom from HNPV to safeguard the life of the patients43. When AVS becomes available for HNPV, these assessments will still be beneficial in clinical decision making. Moreover, in depth analysis of venom cytotoxins will be beneficial in exploring refinements to therapeutic intervention for snake envenoming.Although exact mechanism has yet to be identified, all the HNPV envenomed dogs showed Leukocytosis; which is consistent with As observed in this study, young male dogs, during dusk and dawn, are the most vulnerable group of HNPV envenoming. The clinical sequelae of HNPV bite envenoming in dogs are varied from local to systemic manifestations. Further, venom of this snake may cause life threatening consequences though death due to HNPV bite envenoming in dogs have not been reported. The currently available Indian antivenom is not applicable in HNPV bite treatment but there is a strong rationale to produce antivenom that can neutralize HNPV venom in order to prevent and control the consequences of envenoming by HNPV.


Gastrointestinal helminthiasis poses economic impacts on the dairy sector by reducing milk production. This study aimed at estimating the prevalence of gastrointestinal helminthiasis, the burden of helminths, and appraising potential factors associated with the prevalence in lactating cows. The study was also designed to elucidate the effects of anthelmintic treatment on milk yield, milk fat, and protein content in the Kucha district.A cross-sectional and field clinical trial study designs were used. Standard parasitological techniques (floatation and sedimentation) were employed to detect cows’ infection status. McMaster and Stoll’s egg counting methods were used to estimate helminths' burden. All putative factors that might have been linked with infection were recorded by field observations and farmers' interviews. Sixty cows tested positive for the parasitic infection in the cross-sectional study design were randomly assigned into one of the two groups (dewormed Vs control). Milk yield, milk fat and protein contents were recorded in both groups on day zero and then on weekly basis. Descriptive statistics, binary logistic regression, and repeated measure ANOVA were used to analyze the data.Parampistomum egg, accounting 457.14 ± 275.45 Egg per gram (EPG) of feces. The prevalence of gastrointestinal helminthiasis in cow that had an average body condition score of 1.7 was over two fold higher  than in cows with 6.3 body condition score. A significant improvement in milk yield, milk fat, and milk protein was observed in dewormed cows over 28 days period.Overall, of 422 examined cows, 150  were infected with at least one of the gastrointestinal helminth parasites. Strongyle nematode was the predominant type accounting for 52% of the total record. Analysis of fecal egg count (FEC) in infected cows depicts the highest record of Gastrointestinal helminthiasis is threatening the welfare of lactating cows in Kucha, Ethiopia given its negative association with the body condition score. Gastrointestinal helminths are responsible for the reduction of milk yield and loss of milk fat and protein. However, an improvement in milk yield, milk fat, and milk protein after deworming using Tetrox® (Tetramisole and Oxyclozanide combination), a new drug to the area/ a drug used by relatively few farmers in the study area, proves the effectiveness of strategic deworming.The online version contains supplementary material available at 10.1186/s12917-022-03251-2. The global demand for cow's milk is on the rise. This results from poor production and/or possibly from the ever-increasing world population. Although progressive improvement has been seen in the developing world as regards the production of milk and its products  the demaHaemonchus, Trichostrongylus, Trichuris, Bunostomum, Oesophagostomum, Toxocara vitulorum (T. vitulorum), and others. Gastrointestinal helminth also comprises flatworms, such as Fasciola, Paramphistome, and others flukes [Gastrointestinal helminthiasis is a parasitic infection caused by a group of helminth parasites, which affect the gastrointestinal tract (GIT), associated organs, and whose eggs are excreted to the environment through animals’ faecal material. This group of parasites comprises a variety of species of roundworms, including genus s flukes .Many researchers across the globe have reported a wide range of prevalence for specific gastrointestinal helminth parasites in dairy cows –11. The Ye Kucha Kibe" literally meaning "butter from Kucha" is a name known by the majority of Ethiopians to indicate "high-quality butter". However, a study shows diseases affect butter production in the district [In Kucha, Ethiopia, dairy products, such as milk and butter are considered the major resources base. “district , despitedistrict . GastroiTherefore, the objective of this study was to estimate the prevalence, appraise associated factors of gastrointestinal helminthiasis and to elucidate the effect of anthelmintic treatment on milk quantity, fat, and protein contents in lactating cows in Kucha, Ethiopia.Data on husbandry practices of dairy cow were collected from 422 farmers, which prove 100% response rate. Accordingly, 49% of farmers showed their herd size to be 1–3 dairy cows. The majority of respondents indicated the presence of only one lactating cow (64.2%) with no other domestic animals at home (83%). Over 95% of lactating cow were local Zebu, of which almost 95% being managed extensively. The majority of the cow were young, having an average condition score of 2.6 and at mid-lactation period. The highest proportion of farmers highlighted that they use Albendazole bolus (65.4%), followed by Tetramisole, Ivermectin, and Tetrox (combination of Tetramisole and Oxyclozanide) to lessen the impacts of gastrointestinal helminthiasis  were infected with at least one type of gastrointestinal helminth parasites. The highest prevalence was observed in the multiparous (37.5%); old (43.75%), cow with 1.7 body condition score (47.12%) and cow at late lactation stage (40.51%) that lives in the highland agro-ecology (50%). In addition, slight variation was noted in the prevalence of gastrointestinal helminthiasis in the categories of various explanatory variables, including breed, deworming history, management system and pregnancy status , the majority (68%) had a mono-parasitic infection while only 32% of cows had a poly-parasitic infection. Strongyle was the predominant nematode among mono-infection accounting 52%, followed by Fasciola (11.3%) and Paramphistomum (4.7%). Of the poly-parasitic infected cows, 26.7% had two parasite combinations, particularly Strongyle and Paramphistomum spp. mixture being the dominant group. Moniezia, Trichuris and Ascarid spp. often appeared in mixed form with others or each other, whereas Strongyle nematode detected in all mono- and poly-infections  of faeces, followed by Trichuris (357.14 ± 207.02), Ascarid (320 ± 327.11), Moniezia (290.91 ± 206.26), Strongyle (242.86 ± 205.01) and Fasciola (242.42 ± 169.61)  in infected cows depicts the highest (mean ± sd) record of P = 0.016) than in cows with 6.3 body condition score. Moreover, the prevalence of gastrointestinal helminthiasis in cows, which had 2.6 body condition score was insignificantly higher by 25% compared to the reference group . Meanwhile, there was no adequate evidence of association of infection with other hypothesized factors, such as agro-ecology, age, breed, pregnancy status, deworming history, lactation period, parity and management system.Several host and husbandry factors' relationship with the prevalence of gastrointestinal helminthiasis is given in Additional file P < 0.05) effect of the period (days) and the interaction of treatment and period (treatment*period) on log (x-5). A back-transformed average milk values of dewormed (0.558 L) and control (0.461 L) groups of the cow did not significantly vary (P = 0.9551) on the initial day (D-0) of the trial. Following the initial day (D-0), an average milk quantity steadily climbed in both dewormed and control cows with significant variations (P < 0.05) from week to week until the value hit the peak by 21 days followup time. Average milk value slightly declined without significant change (P > 0.05) thereafter in both groups and remained over 1 L in dewormed cows and under a liter in the control group by the end of the study. The average milk amount of both dewormed and control groups by the end of the study showed a statistically significant gain (+ 0.449 L) and (+ 0.161 L) respectively compared to their initial values (P < 0.001). However, these gains in dewormed (+ 0.449 L) and control (+ 0.161 L) cohorts of the cow are statistically significant (P = 0.049)  effect of period (days) and the interaction of treatment and period (treatment*period) on the square root (x + 5). The initial values of fat content in dewormed (5.06%) and control (5.75%) cow did not significantly vary (P = 0.9809). The fat content of the final day (day-28) in dewormed and control groups was 6.41% and 3.83% respectively. The difference of initial and final values in dewormed group shows an increment (+ 1.35%) however, this gain is not significant (P = 0.126). On the other hand, this difference reveals progressive significant (P < 0.001) loss (-1.92%) in the control group . Accordingly, initial values of milk protein of dewormed (3.17%) and control (3.43%) cows were not significantly different (P = 0.2858). The initial value and the value at the end of the study (3.83%) were statistically significant (P < 0.001) in dewormed animals, however, these values (3.43 Vs 3.53%) did not significantly vary (P = 0.9896) in control cow  anthelmintic drug on the milk yield, milk fat, and protein contents.In the Kucha district, dairy production is one of the methods to generate household income. However, the production system seems suboptimum as many households often keep few number of dairy cows Table . This obFasciola was the most dominant parasite of lactating cows. This difference might be attributed to the variations in the agro-ecology of the region, in which the effect of climatic factors dictate the biology of individual helminth parasites [The overall prevalence of gastrointestinal helminthiasis of lactating cows in the current study could not easily be compared with other studies conducted in either Ethiopia or elsewhere as our methodology was different from many other studies . Nonetheless, we tried to the level we could in order to bring existing literature for the comparisons with our results. In this regards, an overall prevalence in our study corresponds with the prevalence report from Holleta, Ethiopia . Meanwhiarasites .Paramphistomum, which was relatively less prevalent helminth, might suggest the development of resistance against anthelmintic drugs being used in the area. On the other hand, relatively the lower faecal egg count of the most dominant gastrointestinal helminths (strongyle) is interesting. The observed lower egg counts could be due to the presence of a few anthelmintic resistant strongyles, which have a low fecundity profile.The faecal egg estimates for all helminths identified in the current study indicate a mild form of infection . HoweverThe risk factors investigation reveals there was not adequate evidence for almost all hypothesized factors to be significantly associated with gastrointestinal helminthiasis in lactating cow except, body condition score. This implies gastrointestinal helminths infection is equally important in lactating cows irrespective of their difference in host factors, production stages, and husbandry methods. As expected, the significant associations of cows' body condition score and the prevalence of gastrointestinal helminthiasis depict the importance of gastrointestinal helminths in the welfare of dairy cows. The effect of helminthiasis on general conditions of cows possibly due to progressive reduction in body weight  or/and tSignificant variations of milk yield between the dewormed and control cow at the final study period day-28) suggests an improvement of milk yield after intervention against gastrointestinal helminthiasis. Elimination of adult helminths burden by the anthelmintic drug might have reduced stress caused by the parasitism, which contributed to the rise of milk quantity. This finding concords with the report from Nepal , Wales , and Aus suggestsAn improvement of milk fat in dewormed cows whilst a progressive loss of fat content in control cow points to the negative impact of gastrointestinal helminths in the milk fat content. The finding in the Nepal  that illOur report entails a significant improvement of milk protein after deworming the lactating cows. This finding is also consistent with the study from Nepal  and AustIn the cross-sectional study design, we demonstrated an insignificant variation in the prevalence of gastrointestinal helminthiasis between dewormed and non-dewormed cows. Although this condition points the possibility of anthelmintic resistance, we could not be certain to generalize this condition is due to resistance without confirmation through anthelmintic efficacy study. Given an Albendazole being the most widely utilized chemical in the district, in our second study design, we confirmed that strategic deworming  has improved milk yield, milk fat and protein. However, the efficacy of these two drugs (Albendazole versus TETROX 3400) should have been compared in terms of parasitological and dairy production parameters.In conclusion, this study reveals gastrointestinal helminths are threatening the welfare of lactating cows in Kucha district Ethiopia. A comparable infection status in dewormed and non-dewormed cow point to therapeutic failure possibly due to the development of Albendazole resistance given its extensive utilization in the area. However, an improvement in milk yield, milk fat, and milk protein after deworming using Tetrox® (Tetramisole and Oxyclozanide combination), a drug used by relatively few farmers in the study area, proves the effectiveness of strategic deworming (shifting the most widely utilized to seldom used anthelmintic by the farmers). To better explore the effectiveness of shifting anthelmintic compounds on milk yield, fat, and protein, future studies should include both previous and new drugs in the trial. Moreover, we encourage the study on a field anthelmintic efficacy test in lactating cows in the Kucha district.Kucha is one of the districts in the Southern Nations, Nationalities, and Peoples' Region (SNNPR) of Ethiopia Fig. . The altThe survey was conducted in the period between January 2021 to March 2021 to execute the aforementioned objectives. The study animals comprised randomly selected lactating cows with different characteristics that were reared under an extensive and semi-intensive management system by smallholder farmers. As recruitment criteria, all lactating cows at different lactation stages, management systems, and physiological states in the Kucha district were selected for enrolling in the study. However, cows under severe illness and those cows whose owner was not willing to give consent for participation were excluded from the study.The required number of lactating cows for the cross-sectional study was calculated using the formula given below , by consn = 1.962*Pexp (1-Pexp)/d2 Where n = sample size, Pexp = Expected prevalence, d = desired absolute precision (5%), and 1.962 = z-value at 95% confidence level.A stratified random sampling  techniquBefore taking a biological sample, information related to the individual animal, such as breed, age, parity, stage of lactations, pregnancy status, body condition score, and management-related data, including production system, deworming history within the last 4 weeks were obtained through household interview in local language using a structured questionnaire format  of faeces for positive cows. The McMaster technique was developed at the McMaster laboratory of the University of Sydney and first published in 1939 in the journal of the council for scientific and industrial research by Gordon and Whitlock. Since its first description, many modifications for the technique have been reported. Today, traditional McMaster technique uses two section-counting chamber, which enables to estimate eggs/oocyst from a known volume of fecal suspension by microscopic examination. Briefly, 3 g of faeces was thoroughly mixed in 42 ml of saturated sodim chloride solution (specfic gravity ~ 1.2). The mixture was poured through the kitchen sieve into a laboratory beaker. Aliquates were transferred and filled in to both chambers of McMaster slide uing plastic pasteur pipete. After filling the chambers, the slide was allowed to sit for 5 min. Eggs in both grids of chambers were counted, sumed up and multiplied by 50 to get the number of eggs in a gram of faeces .Stoll’s techniques was applied in order to estimate fluke burden as fluke eggs do not float in sodium chloride solution. A 3 g of feces was emulsified in 42 ml of tap water. The mixture was poured through the kitchen sieve into a laboratory beaker. After that, the solution in the beaker was further strained through a 150 µm laboratory sieve into the conical tube. The tube was allowed to stand for about 3–5 min at room temperature. The supernatant was siphoned off and a drop of 1% methylene blue was added to the sediment. After mixing gently, using a micropipette, 0.15 ml of sediments were transferred to two microscopic glass slides, covered with a cover clip and finally examined under microscope at 100 magnification. The number of egg in both slides were counted, summed up, and multiplied by 50 to get fluke egg per gram (EPG) of faeces as described for McMaster technique .P-value at 5%, and a study power of 80% were considered by assuming an equal number of animals in the groups. Online epidemiological calculator Epitools (Ausvet®) was used for sample size calculation [The study population for field clinical trial comprised lactating cows infected with gastrointestinal helminths based on the first cross-sectional study. The required sample size for field clinical trial was calculated from the hypothesis that the mean milk protein content difference between anthelmintic treated and untreated lactating cows was 0.105% from the previous study . For thiculation . AccordiN = 60) were local Zebu, of which half (N = 30) were multiparous at early lactation phase while another half (N = 30) were at the second parity and at mid-lactation stages. The body weight of cow was estimated using heart girth measurement. An average body weight of cow was 218.5 kg. Cows in each stratum were randomly assigned into one of the two treatment groups (dewormed and control). Cows in the dewormed group were drenched a bolus that consisted of Oxyclozanide and Tetramisole hydrochloride  at the dose rate of 22.6 mg/Kg of body weight in a single dose. This anthelmintic was chosen over others due to its relatively low frequency of utilization by the framers in the area , and allowed to exercise for at least 2 h outside the shelter once in a week.Farmers were instructed to manage their cows indoor until the end of study period. For all cows, the basal diet consisted natural grass hay. Dairy concentrate feed composed of wheat bran; noug seed cake and common salt was given at the rate of 0.5 kg/L of milk every morning on the daily basis. They were also fed locally available vegetable leftover, such as air-dried chips of sweet potato and cassava roots (Manihot esculenta Crants) at the rate of 1 kg/week. Traditional homemade alcoholic product, “The volume of milk yield of the individual cow in both treatment groups was recorded five times in a month  using a measuring cylinder during milking. To determine milk protein and fat contents, 10 ml milk sample was collected in a sterile vial on the same days mentioned above. The sample was then, placed in an Ice Box that contained an ice pack for transportation to the dairy technology laboratory of Arba Minch University, College of Agricultural Science. The fat and protein contents of milk collected from each cow were analyzed using an automated Lactoscan milk analyzer . The protocol was employed as per the manufacturer's directions.The information generated from the study was recorded in hard copy format before being prepared in an electronic Microsoft excel sheet. The number of infected cows with at least one type of helminth parasite among total examined cows, the magnitude of each gastrointestinal helminth from a total positive cow, and epidemiological factors were presented in proportion and 95% confidence intervals for proportion using exact binomial test. Mean ± sd was used to present the burden of individual helminths parasites in the positive cow. The association of different hypothesized risk factors, such as agro-ecology, age, parity, lactation period, pregnancy status, deworming history, management system, and breed with overall gastrointestinal helminthiasis (dependent variable) was tested using the binary logistic regression. Odds ratio was calculated to estimate the degree of the associations. P-value and 95% confidence interval for odds ratio were used to judge the statistical significance.The impact of deworming on milk parameters was analyzed using repeated measure ANOVA in a linear mixed regression model. The models were run using the lmer  function from “lmerTest” package. The models included the main effects (deworming and observation time), their interaction term, and random error . The adjusted average values of milk yield, fat, and protein content were estimated using the function emmeans  from “emmeans” package. Contrast  function was used for post-hock pairwise comparisons in Bonferroni method. The values were presented in the least Squares Means ± Standard Error (LSM ± SE) using a line graph in ggplot  function from “ggplot2” package. For all analysis, 5% marginal error, and 95% confidence level was considered. The R program version 4.1.0.  was usedThe preliminary analysis showed that the residual error of milk quantity (L), protein (%), and fat (%) content were not normally distributed when assessed using graphically (Q-Q plot)  and analysis of risk factors using univariable binary logistic regression in Kucha, Ethiopia.Additional file 2: Supplementary Figure. Schematic representation of the sampling procedure of the study subjects in Kucha, Ethiopia.Additional file 3: Supplementary format. Data collection format to investigate animal-level and husbandry factors on overall prevalence.Additional file 4: Supplementary others. Milk yield, milk protein and fat contents data analysis outputs using R-program.


In contrast, lengthier fair to vigorous activity and sleep durations decreased the odds of a VA event (p < 0.001). Future studies using consumer wearables in a larger population should prioritize these outcomes to further assess VA risk.Ventricular arrhythmia (VA) is a leading cause of sudden death and health deterioration. Recent advances in predictive analytics and wearable technology for behavior assessment show promise but require further investigation. Yet, previous studies have only assessed other health outcomes and monitored patients for short durations (7–14 days). This study explores how behaviors reported by a consumer wearable can assist VA risk prediction. An exploratory observational study was conducted with participants who had an implantable cardioverter-defibrillator (ICD) and wore a Fitbit Alta HR consumer wearable. Fitbit reported behavioral markers for physical activity , sleep, and heart rate. A case-crossover analysis using conditional logistic regression assessed the effects of time-adjusted behaviors over 1–8 weeks on VA incidence. Twenty-seven patients  were included. Among the participants, ICDs recorded 262 VA events during 8093 days monitored by Fitbit (median follow-up period 960 days). Longer light to fair activity durations and a higher heart rate increased the odds of a VA event ( Heart arrhythmias constitute a growing challenge to healthcare systems worldwide, and ventricular arrhythmias (VA) are an important and increasingly frequent cause of sudden death and health deterioration. Implantable cardioverter-defibrillators (ICDs) and remote monitoring have led to significant advances in reliably avoiding malignant VAs ,2, leadiDuring the last decade, there has been a significant increase in the availability and use of health monitoring devices and mobile applications that provide users with personalized health data. The reasons for the use of such tools are diverse and include the desire for behavior change or health monitoring among healthy users as well as users with chronic diseases, such as cardiovascular disease ,7. ConsuThis gap in the literature motivates further research into the usefulness of wearable activity trackers for heart arrhythmia risk prediction, and there is a specific need to conduct studies with prolonged periods of activity data collection. Based on prior studies, we hypothesize that consumer-wearable activity trackers represent an opportunity for improving early risk prediction of VAs and can potentially support early clinical intervention. We specifically aim to examine general behaviors among a patient population with ICDs over an extended period  and thereby identify relationships between daily behaviors and VA events.An exploratory observational study was conducted to understand how VAs among patients with ICDs may be associated with behavioral data preceding a VA event, as collected from a consumer-wearable activity tracker. This study was part of a more extensive research and development project, SCAUT , conducted from 2014 to 2018, which aimed to improve early detection of deterioration in patients with a cardiac device and communication between such patients and health professionals ,18,19,20This study comprised a sample of 27 patients with a secondary prevention ICD, a device that is offered to individuals who have survived sudden cardiac arrest or who have a history of VAs . PatientAs part of the SCAUT research and development project, this study was conducted according to the guidelines of the Declaration of Helsinki, approved by the Danish Data Protection Agency and reviewed by the Capital Region of Denmark’s Regional Committee for Health Research Ethics (No. H-19029475). Informed consent was obtained from all subjects involved in this study. Patients were informed that the Fitbit device is a consumer-wearable activity tracker and not a clinical device, and that it reports data without diagnostic validity.TM Report Export [The first data source was the ICDs, which provided data report files on remote heart rhythm monitoring in XML format through Medtronic CareLink . Three tt Export : ventricThe second data source was the Fitbit Alta HR  consumer wearable activity tracker. The Fitbit data were collected through an application programming interface  that proThe Fitbits counted participants’ steps and classified the physical intensity as sedentary, light, fair, or vigorous for each 15-min interval in a day . For time periods of assumed sleep, the Fitbits classified the sleep type as asleep, awake, restless, or unknown for 1-min intervals . Fitbit did not provide precise thresholds for its physical activity recognition algorithms . Thus, iThe Fitbit variables were aggregated for analysis. The variables were first aggregated over days, then weeks, then intervals of 1–8 consecutive weeks called periods. The reason for deriving different periods was to explore the risk of VA events for the purposes of timely clinical intervention . Inferential and descriptive analyses using the aggregations were then conducted. The data analysis was performed in Python  using thFitbit measurements reported as “0” were excluded from analysis. Valid days, weeks, and periods were then derived according to several scenarios. First, only days with at least one, two, four, or eight hours of physical activity data available  between 8 a.m. and 8 p.m. were deemed valid days and included in the analysis as four separate scenarios. Second, only weeks with at least four, five, or seven valid days were included as valid weeks as three separate scenarios. Third, only periods with sufficient valid weeks were deemed valid periods according to three increasingly strict scenarios: minimum 50%, 75%, and 100% valid weeks . This syData aggregation was conducted through accumulation of all variables from the 15-min intervals in valid Fitbit days. Steps, physical activity duration, and sleep duration were summarized via daily aggregation to support the subsequent analysis. This approach has also been implemented in other studies using Fitbit data ,40,41. DThe descriptive analysis consisted of two parts. The first part concerned summary statistics  for data quality and behavioral markers. The second part concerned changes in Fitbit wear in the temporal vicinity of VA events observed for individual patients. For brevity, only the first part of the descriptive analysis is included in this paper. The second part is detailed in The inferential analysis assessed the extent to which the given behaviors affected the odds of a VA event over time. This assessment was performed by means of a case-crossover design using conditional logistic regression ,43. ThisFor each patient, all monitoring days were used to capture the outcome of VA or no VA on any given day. Windows of valid Fitbit-measured periods of 1–8 weeks were used to define the exposure immediately succeeding each day of VA (case periods) or no VA (control periods). continuous exposure) and (b) time-specific variables for time-point adjustment: (i) season , (ii) day of week (1–7), and (iii) weekday status  of the date immediately succeeding the period. A scenario defines a specific combination of predictors: (a) behavior aggregate variables and (b) time-specific variables. Three conditional logistic regression formulae were derived. A total of 108 scenario-formula combinations resulted from 36 scenario combinations and three formulae  affected the change in probability of a VA event. The predictors in the conditional logistic regression models were (a) behavior aggregate variables  and for periods of durations in weeks at most the fixed duration 1–8 weeks, falling within the larger duration scope (denoted as combined models), as illustrated in p < 0.001) were expected. If, for a given behavior, across all models, (1) there were no significant ORs or (2) some significant ORs were sub-unit and some significant ORs were supra-unit, the OR was reported as inconclusive.As the objective was to explore patterns without focusing on individual results, any odds ratio (OR) that exceeded the significance threshold ɑ = 0.05 was reported, without adjustments for multiple comparisons. However, highly significant ORs  who were invited to participate in the study, 27 participants provided written informed consent. Of these, 25 were male (93%), and the median age among participants was 59 years (mean 57.3 ± 11.1), as presented in VA events were reported in 16 of the 27 participants. A total of 262 different types of VA events were recorded, with a mean of 16.4 ± 31.7/patient over a mean duration of 32.5 ± 28.9 months/person. Of these events, 56 were ventricular tachycardia , 172 were ventricular tachycardia type 1 , and 34 were ventricular fibrillation and ventricular tachycardia (VF-VT) events (mean 2.1 ± 2.3 events/patient). The VA events by type for each patient are presented in Fitbit-recorded behavioral data were available for all 27 patients with a total of 11.769 days . The median ICD follow-up period was 960 days (mean 991.3 ± 880.9 days/patient). The follow-up periods for each patient are presented in As previously mentioned, Fitbit-recorded behavioral markers for physical activity, sleep, and heart rate were collected for the 27 patients. Mean daily physical activity consisted of 7667.7 ± 3521.6 steps; 352.8 ± 89.8 min/patient in light activity duration; 43.4 ± 40.1 min/patient in fair activity duration; and 60.6 ± 33.0 min/patient in vigorous activity duration. For heart rate, the Fitbits recorded a mean of 50.3 ± 6.7 beats/min for the daily minimum, 69.3 ± 9.8 beats/min for the daily mean, a median mean of 66.2 ± 10.7 beats/min, a maximum mean of 124.9 ± 12.7 beats/min, and SD mean 4.2 ± 0.9 beats/min. More details are available in Increases in light to fair physical activity duration and in heart rate resulted in an increased risk probability of a VA event; conversely, increases in fair to vigorous physical activity duration and sleep duration that included the awake sleep type  resulted in a decreased risk probability of a VA event .Spending more time in light to fair physical activity increases the risk of VA events. On average, the odds increase by 9 to 20 percent when time spent in light-intensity physical activity increases by 15 min per day, as measured over 1–3 weeks. Furthermore, 15 additional minutes of light or fair activity led to an average odd increase of 9 to 12 percent, as measured over 1–2 weeks.However, fair to vigorous activity reduces the risk of VA events. The odds decreased by 32 to 34 percent on average with every 15 extra minutes of fair activity per day, measured over 4–8 weeks. The odds also decreased by 16 to 21 percent on average with every 15 extra minutes of vigorous activity, measured over 2–8 weeks. Furthermore, 15 more minutes of combined fair and vigorous activity reduced the odds by 12 to 16 percent, as measured over 2–8 weeks.A higher heart rate increases the risk of VA events. Ten extra beats per minute increase the odds of a VA event as follows: minimum heart rate measured over one week doubled the odds, mean heart rate measured over 2–3 weeks increased the odds four to ten times, median heart rate measured over 2–4 weeks increased the odds four to sixteen times, and maximum heart rate measured over two weeks doubled the odds of a VA event. More findings are available in This exploratory observational study assessed the relationship between behavioral activity changes and the risk probability of potentially life-threatening VA events by comparing data from ICDs and Fitbit wearable activity trackers. We found that higher heart rates and spending more time in light to fair physical activity increased the risk of imminent VA events, whereas fair to vigorous activity reduced the risk. Few previous studies have assessed the risk probability of VA events using technology-reported data from ICDs and wearable activity trackers, especially with longer follow up times. By assessing the utility of consumer-grade wearable activity trackers for early risk assessment of VA events, the aim is to build towards the validation of interpreting significant behavior changes  as a vital clinical sign for early clinical intervention among patients at risk of life-threatening heart arrhythmias.Our cohort was representative of an ICD population with regards to age and gender, with a predominance of males. The results indicated that increased duration of light or light + fair physical activity increased the risk probability of a VA. Conversely, a decrease in fair, fair + vigorous, or vigorous activity levels increased the risk probability of a VA. Our results therefore support a cardioprotective effect of exercise  and suggBased on our findings, future studies could measure light physical activity over shorter time intervals (1–3 weeks). They may consider 9 percent as a baseline odd increase for 15 extra minutes of light to fair activity. Fair and vigorous physical activity could be measured between 2 and 8 weeks, where future studies may consider the average odds decreases of 32% and 16% for every 15 extra minutes of these activity intensities. Heart rate yielded significant effects within 1–3 weeks of monitoring. Ten extra beats per minute increased the odds twofold on average. Heart rate could be monitored closest, as changes associated with the minimum and maximum heart rates were visible in short periods .This study does not report key findings related to sleep. This choice was made because we noted that Fitbit may not always have reported sleep durations for the patients, as the awake, sleeping, and non-wear times were accumulated in the dataset. Nevertheless, prior literature has described an association between sleep behavior and physical activity for patient-reported physical function, quality of life, and cognitive function, though often with the limiting factor of self-reported sleep outcomes in homogeneous populations ,50. As tThe results from this study and similar previous studies suggest that several critical aspects may influence the quality of data collected and pose potential scaling issues when leveraging consumer-wearable activity trackers for VA risk assessment among ICD patients. Although the results are indicative, they suggest that consumer-wearable activity trackers can be leveraged for VA event risk assessment, potentially enabling better (self-) management of activities contributing to health , and may ultimately lead to improved health outcomes.There are several implications for systems designed for risk prediction of VAs. First, there is, on the one hand, the potential derived from using external research-grade wearable activity trackers, which can capture high-granularity and high-accuracy data . Such deFor patients who accept consumer-wearable activity trackers, the accuracy of the predictive performance and the timeliness of notifications are critical for the success of usage and collected data quality . In an eA strength of this study is that it is the first to examine the outcome of VA, using raw data from ICDs and compare it to data from a consumer-wearable activity tracker. Second, the activity tracker wear time was on average over one year, which far exceeds the average wear time in previous studies . Third, p < 0.001). These results would therefore remain visible with statistically significant corrections up to 50×. Furthermore, patient-effect was adjusted for by means of distinguishing each behavior-time-event data point as unique to the patient, and no additional variables could be added, avoiding collinearity.This study has several limitations. First, the small sample size of unique patients prevents a separate analysis based on age or gender and limits our confidence regarding the generalizability of the findings to a larger population. The results are based on a large amount of longitudinal data with several time epochs per individual patient. This approach poses a risk of bias by carry-over, but arguably resulted in a conservative analysis given that any association between exposure and outcome had to be robust to nullify inverse or null associations during parts of the same time epoch. We allowed a permissive significance threshold of ɑ = 0.05 without adjustments for multiple tests, but also found highly significant results  and sleep . In addition, Fitbit did not distinguish sedentary duration as time awake, sleeping, or non-wear. This limitation made it difficult to delineate and thereby analyze these behaviors and may explain the mean recorded sleep duration of under four hours. This limitation was accounted for by excluding the sedentary duration from the analysis.Third, feedback about behaviors  and observations reported to patients by the Fitbit device and associated application might have influenced the behaviors under study. The patients may have changed their physical activity patterns, or sleep patterns based on the feedback provided by the device.Finally, the patient data did not contain baseline characteristics—such as concurrent heart disease, presence of heart failure, medications, or comorbidities —that may be confounders, influencing the behavioral, as well as the VA outcomes.In the light of the increased availability and reliability of consumer-wearable activity trackers, this study explored the extent to which daily behaviors reported by such trackers can assist in VA risk assessment in ICD patients. The results indicated that increased levels of activity are cardioprotective, decreasing the odds of experiencing a VA event. Future studies using consumer-wearable activity trackers in a larger population can further refine our findings to assess the risk of VA.


Arabidopsis thaliana, but studies in other species have not consistently shown the benefits of photons in the green region on leaf expansion and growth. Spectral effects can interact with total photon flux. Here, we report the effect of the fraction of photons in the blue (10 to 30%) and green (0 to 50%) regions at photosynthetic photon flux densities of 200 and 500 µmol m−2 s−1 in lettuce, cucumber and tomato. As expected, increasing the fraction of photons in the blue region consistently decreased leaf area and dry mass. By contrast, large changes in the fraction of photons in the green region had minimal effects on leaf area and dry mass in lettuce and cucumber. Photons in the green region were more potent at a lower fraction of photons in the blue region. Photons in the green region increased stem and petiole length in cucumber and tomato, which is a classic shade avoidance response. These results suggest that high-light crop species might respond to the fraction of photons in the green region with either shade tolerance (leaf expansion) or shade avoidance (stem elongation).The photon flux in the green wavelength region is relatively enriched in shade and the photon flux in the blue region is selectively filtered. In sole source lighting environments, increasing the fraction of blue typically decreases stem elongation and leaf expansion, and smaller leaves reduce photon capture and yield. Photons in the green region reverse these blue reductions through the photoreceptor cryptochrome in  In naturally shaded environments, irradiance/photon fluxes in the blue (400 to 500 nm) and red (600 to 700 nm) regions are relatively reduced while the fluxes of photons in the green (500 to 600 nm) and far-red (700 to 750 nm) regions are relatively enriched. In the photobiology literature, the term light is often used to refer to the photon flux, as in blue light, but this terminology does not describe the discrete nature of photons, which drive photobiological reactions. Additionally, light is closely connected to brightness in human perception of photons, thus photon is a preferable term. Here, the terms blue, green, red and far-red photons refer to photons in the regions that induce blue, green, red or far-red color perception.Plant developmental responses to a relative increase in far-red have been well studied ,2, with Elevated far-red commonly increases leaf area in controlled environments and this response is beneficial because it increases photon capture . IncreasLactuca sativa cv. “Waldmann’s Green” by 31% and increased shoot dry mass by 47% [Early light-emitting diode (LED) fixtures for horticultural applications supplied only blue and red photons. One of the first studies that investigated the effects of adding green to this type of spectrum found that increasing the fraction of green photons from zero to 24% increased leaf area in s by 47% . This eas by 47% ,7. Howevs by 47%  , suggesArabidopsis thaliana mutants have indicated that cryptochromes are the primary photoreceptor influencing the decrease in stem length [Cryptochromes are one of the two most well-studied families of blue photon receptors, and they primarily modulate plant growth through the control of gene expression. The other well studied family of blue photon receptors are phototropins, which primarily modulate plant growth through interactions with membranes ,10. Sunlm length ,12. Longm length . Phototrm length , but thiArabidopsis extend to these horticultural species. We review 29 studies spanning 19 years on the effects of blue photon fraction in lettuce, cucumber and tomato in Studies have typically found that increasing the fraction of blue photons decreases leaf area/plant diameter in the horticultural crops lettuce ,22,23,24o), which is the active form. Photon absorbance by FADHo induces conversion into the fully reduced (FADH–) state, which is inactive [o shows relative lower absorbance in the blue region and higher absorbance in the green region [– by photon absorbance [Unlike spectral distributions that lack either blue or red photons ,26,43, ginactive ,45. The n region . The fuln region ,48, but sorbance ,49. ThisArabidopsis thaliana [This hypothesis has been evaluated in multiple studies. Although increasing the green photon flux can induce shade morphology  in thaliana ,51, the thaliana  many oththaliana ,27,29,33thaliana , which iAdditionally, replacing red photons with green photons under a constant fraction of blue has increased leaf area and dry mass in some studies ,39,41,52The photosynthetic photon flux density (PPFD), or intensity, affects morphology. One notable example is that leaf thickness typically increases with increasing photon flux. Thick and thin leaves are referred to as sun and shade leaves. This response has recently been partially explained by the involvement of both cryptochromes and phototropins . In somePrevious studies have investigated some of the following interactions: (1) the effect of blue photons between 10 and 30% blue ,26, (2) Lactuca sativa, var. Red Salad Bowl), tomato  and cucumber  seeds were direct seeded then thinned for uniformity after emergence leaving four plants per module. Planted root modules were randomly placed into the 16 treatment chambers. Each chamber had dimensions of 20 × 23 × 30  with gloss white walls. Fans provided an air velocity of 0.5 m s−1 at the top of the canopy. The root modules measured 20 × 18 × 13 (4680 cm3) and contained a 1:1 ratio of peat and vermiculite by volume with five grams of uniformly mixed Nutricote ® slow-release fertilizer . Root modules were watered to 10% excess as needed with dilute fertilizer solution , and were allowed to passively drain. Type-E Thermocouples connected to a data logger  continuously monitored ambient air temperature at the top of the plant canopy. Day/night temperature was 23/20 °C, with less than 1 °C variation over time and 1 °C variation among chambers. CO2 concentration was continuously monitored and was identical for all treatments and varied over time between 450 and 500 ppm.Lettuce . Treatments were developed using LEDs  to output three white , three red/blue (RB) combinations, and two red/blue/green (RBG) combinations. The RB combination had about 10, 20 and 30% blue, and the RBG treatments contained about 10 and 20% B with 20 or 10% G, respectively. The spectral distributions of the treatments were measured before each replicate study with a spectroradiometer  and are shown in The system included 16 chambers with eight unique spectral outputs at two intensities for a 16 h photoperiod (PPFD: 200 µmol m2leaf m−2ground) was calculated by dividing total leaf area per chamber by the ground area of the chamber. Shoot dry mass (DM) was measured after the tissue was dried at 80 °C for 48 h. Dry mass per unit area (g DM m−2ground) was calculated by dividing total dry mass by the chamber area. Specific leaf mass  was calculated by dividing the total leaf dry mass of the four plants by the total leaf area of the four plants. The average stem and longest petiole length from each chamber were used for statistical analysis (four measurements averaged together).All species were harvested after canopy closure – when the leaves of the four plants in one of the treatments began to touch. This occurred 21 days after emergence in lettuce, 12, 13 and 20 days after emergence in tomato, and 11 or 13 days after emergence in cucumber. At harvest, stem and longest petiole length of the each of the four plants per chamber were measured in tomato and cucumber. Leaf area was measured using a leaf area meter . Leaf area index . In a mixed effects linear model, percent blue and percent green were treated as continuous variables while intensity was treated as a fixed effect. Replicates were treated as random factors. Interaction terms between these three factors were included in the linear model. The three-way interaction was insignificant for all parameters and was therefore pooled into the error term.The study was replicated three times (each with four plants per replicate in time). All data were analyzed using R statistical software . Blue, green and PPFD effects on the growth parameters in lettuce, cucumber and tomato were determined using lmer and Anova functions with an F statistic. We present significance at −2 s−1). This separation was also done in the analysis of the effects of green photons, and because green photons have been implicated in the reversal of blue photon effects, the data were further separated for 10 or 20% blue photons. The RB30 treatment was not included in this analysis, and both the cool and neutral white LEDs were considered about 20% blue.In order to understand the interactions, the effect of blue photons was also analyzed by separating the data by intensity (200 and 500 µmol mRepresentative photos of the three species in each treatment are shown with the percentages of blue and green in −2 s−1).Significant effects from the mixed effects linear model are presented in −2 s−1) resulted in an increased dry mass in all three species  compared to the lower intensity (PPFD = 200 µmol m−2 s−1) in all three species . These two chromophores (FAD and FMN) are structurally very similar, and as such they have similar absorbance properties for each of their oxidized, semi-reduced and fully reduced states . For cryve state ,45,61. Dve state . In a sive state , the revve state . Nonetheve state , and thuIn this study, both lettuce and tomato showed a significant decrease in SLM with an increasing fraction of green photons d,f. ThisPhotoreceptors may be saturated at higher photon fluxes, meaning that spectral shifts at higher intensity may have a smaller impact on plant morphology than at lower intensities. By contrast, preferential absorption of blue photons by pigments other than the blue photon receptors may minimize plant responses to lower intensities of blue photons. The latter scenario may be the case for the interaction between percent blue and PPFD in tomato SLM, as increasing percent blue at the higher PPFD increased SLM by 16%, but appeared to have no effect at the lower PPFD c. The inDespite these perplexing interactions, the overall response of SLM to an increase in the fraction of blue and green photons is generally consistent with the role of cryptochromes and phototropins.Mechanisms underlying leaf area are complex due to interactions with photosynthesis. Here, we review the potential role of phototropins and cryptochromes on the control of leaf expansion under blue and green photons.Arabidopsis thaliana mutants lacking phototropins have smaller curled leaves compared to wild-type plants [In a young seedling, blue photons contribute to early cotyledon expansion, and both cryptochromes and phototropins are involved in this response ,12. Matue plants ,66,67. TCryptochromes have been implicated in low blue shade avoidance responses like hyponasty , but thecry1cry2phot1phot2). Altogether, increasing the fraction of blue photons (perceived by cryptochromes) may both increase periclinal cell division at the expense of anticlinal cell division, and increase cell expansion along the abaxial to adaxial axis at the expense of elongation along the perpendicular axis. Considering the potential role of cryptochrome and phototropin in SLM, it follows that increasing leaf thickness through cell elongation in the abaxial to adaxial direction may decrease leaf expansion. Dougher and Bugbee  found thAlthough the mechanisms underlying the effect of blue photons on leaf expansion are not clear, cryptochromes appear to modulate the response. It is difficult to determine the role of phototropins in leaf expansion. Further research is warranted.The decreases in dry mass as the fraction of blue photons increased were likely caused in part by the lower quantum yield of blue photons compared to green and red photons. McCree  showed tIncreasing the fraction of blue photons decreased LAI in all three species, while increasing the fraction of green photons only increased LAI in tomato f. As dryDespite the early findings of Kim et al. , who shoArabidopsis thaliana, but the shade avoidance responses of stem and petiole elongation in horticultural species are often absent [Green photons have been shown to induce shade avoidance responses in n absent ,27,29,33n absent ,26,28,332 mol−1 for the activation of cry2 by 450 nm blue photons and 30 m2 mol-1 for de-activation by 560 nm green photons [The interactions between blue photons and green photons in predicting leaf area of lettuce and cucumber, and petiole length in cucumber can be explained by the sensitivity of cryptochrome to blue and green photons. Bouly et al.  measured photons . Photoco photons ). The re photons  indicateGreen photons only benefitted tomatoes. These data are contrary to the early findings of Kim et al. , who fouWe define shade tolerance as an increase in leaf area in response to shade-like light, and shade avoidance as an increase in stem elongation. Based on these definitions, tomatoes expressed both shade avoidance and shade tolerance in response to an increased fraction of green photons, while cucumber only exhibited shade avoidance.Blue photons typically decrease leaf area and dry mass. It therefore seems advantageous to include a low fraction of blue photons in a fixture. From our data, the lowest percentage of blue (10%) produced the highest dry mass. We are now studying the minimal amount of blue needed for normal plant growth.