GSTe2), we established that GST-mediated resistance is reducing the efficacy of LLINs against Anopheles funestus. Hut trials performed in Cameroon revealed that Piperonyl butoxide (PBO)-based nets induced a significantly higher mortality against pyrethroid resistant An. funestus than pyrethroid-only nets. Blood feeding rate and deterrence were significantly higher in all LLINs than control. Genotyping the L119F-GSTe2 mutation revealed that, for permethrin-based nets, 119F-GSTe2 resistant mosquitoes have a greater ability to blood feed than susceptible while the opposite effect is observed for deltamethrin-based nets. For Olyset Plus, a significant association with exophily was observed in resistant mosquitoes . Furthermore, GSTe2-resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0  and PermaNet 3.0 (side)  but not for Olyset Plus. This study shows that the efficacy of PBO-based nets  against pyrethroid resistant malaria vectors could be impacted by other mechanisms including GST-mediated metabolic resistance not affected by the synergistic action of PBO. Mosaic LLINs incorporating a GST inhibitor  could help improve their efficacy in areas of GST-mediated resistance.Growing insecticide resistance in malaria vectors is threatening the effectiveness of insecticide-based interventions, including Long Lasting Insecticidal Nets (LLINs). However, the impact of metabolic resistance on the effectiveness of these tools remains poorly characterized. Using experimental hut trials and genotyping of a glutathione S-transferase resistance marker (L119F- GSTe2). This marker confer metabolic-mediated resistance to pyrethroids and dichlorodiphenyl-trichloroethane (DDT) in the major malaria vector Anopheles funestus in West and Central Africa [GSTe2) has been associated with DDT and/or pyrethroids resistance in several mosquito species including An. gambiae [An. funestus [Aedes aegypti [An. funestus, genomic and structural analyses revealed that a leucine to phenylalanine amino acid change (L119F) in GSTe2, has enlarged the substrate-binding pocket of the enzyme, conferring DDT/pyrethroid resistance in West and Central African populations [The scale-up of insecticide-based interventions including Long Lasting Insecticidal Nets (LLINs) and Indoor Residual Spraying (IRS) has significantly contributed to the considerable reduction of malaria burden in the past decade . Unfortul Africa . Besidesl Africa  either tl Africa . Over-ex gambiae , An. funfunestus  and Aede aegypti . In An. ulations . This siulations  providinAn. funestus across Africa is driven by metabolic resistance [(kdr) has been reported so far for this species [An. funestus through over-expression of detoxification genes such as GSTs or P450s makes this vector suitable to assess the impact of metabolic resistance on control interventions. The presence of the GST-mediated metabolic resistance in An. funestus populations such as in Cameroon [The pyrethroid resistance in sistance ,14,15 as species . This prCameroon ,17,18 prkdr [Experimental hut studies are the method of choice to evaluate the efficacy of LLINs against mosquito populations using entomological indices as a proxy for potential epidemiological impact ,20. It akdr .An. funestus population from Cameroon using experimental huts. Furthermore, we took advantage of the L119F-GSTe2 DNA-based diagnostic assay [Here, we assessed the performance of conventional pyrethroid-only nets versus PBO-based against a pyrethroid resistant ic assay , to assePlasmodium falciparum but also P. malariae [An. funestus s.l. Human activities are mainly fishing, hunting and subsistence farming including maize, watermelon and coffee plantations. At the experimental station, 12 huts built following the World Health Organisation (WHO) standard [An. funestus s.s. was the main malaria vector in the area [Mansonia sp., and Culex sp. were also present in the area. Anopheles mosquitoes in the area were highly resistant to pyrethroid and DDT [The experimental hut station was located in Mibellon , a village in Cameroon located in the Adamawa Region; Mayo Banyo Division and Bankim Sub-division. The Adamawa region is in the mountainous zone forming a transition between Cameroon’s forested south and savanna north. Malaria transmission is perennial with a high transmission as shown by very high infection rate of Plasmodium infection caused by malariae . The vilstandard , were avthe area . Mansoni and DDT . The triThe huts are built following the prototype recommended by WHO for the West African region . The hutDuring the experimental hut trial, four LLINs and one untreated net as negative control were compared . These iPrior to the study, the hut effect was assessed to evaluate any specific attractiveness of huts. Untreated nets were hung in the six huts used for the study, and during 3 weeks, between the 10 and 29 May 2016, volunteers sleeping underneath collected mosquitoes each morning.An. coluzzii susceptible Ngousso laboratory strain. This was done to confirm the quality of the five bed nets used in the study. Cone bioassays were also done in the insectary using F1 progeny from field collected Anopheles funestus from Mibellon. For PermaNet 2.0, PermaNet 3.0 (side and top), Olyset and Olyset Plus beside an untreated control net, five batches of 10 unfed females, 2–5 days old, were exposed to each bed nets using WHO cone assays for three minutes [GSTe2 genotypes and resistance to these insecticides.Cone bioassays were performed at the beginning of the study using the  minutes . They weThe experimental hut trial was carried out during 60 night following the protocol described in the guidelines for laboratory and field-testing of long-lasting insecticidal nets . To corrMosquitoes were collected every morning, using hemolysis tubes from: (i) inside the nets, (ii) in the room: floor, walls and roof, and (iii) in the veranda exit trap. Mosquitoes collected from each compartment were kept separately in a bag to avoid any mixing between samples from different compartments. Samples were then classified as dead, alive, blood fed or unfed. The ‘alive’ samples were kept in the paper cup and provided with sugar solution for 24 h and mortalities monitored.An. funestus group were species identified to species specific level using a cocktail PCR as previously described [The field-caught females were sorted according to morphological keys as previously described . Mosquitescribed .Deterrence/entry rate: the reduction in hut entry relative to control. Deterrence (%) = 100 × (Du − Dt)/Du, where Du is the total number of mosquitoes found in untreated hut (control) and Dt is the total number of mosquitoes in the treated hut.Entry rate (%) = 100 × (Ht/Hn) where Ht is the total number of mosquitoes found in the hut and Hn is the total number of mosquitoes collected in all the 5 huts.Exophily (Excito-repellency): the proportion of mosquitoes found exited in the veranda trap Exophily (%) = 100 × (Ev/Et) where Ev is the total number of mosquitoes fund in veranda and Et is the total number of both inside the hut and veranda.Blood feeding rate (BFR). This rate was calculated as follows: Blood feeding rate = (N mosquitoes fed) × 100/total N mosquitoes. Where “N mosquitoes fed” was the number of mosquitoes fed, and “total N mosquitoes” was the total number of mosquitoes collected.Blood-feeding inhibition (BFI): the reduction in blood-feeding in comparison with the control hut. Blood feeding inhibition is an indicator of personal protection (PP). More precisely, the personal protection effect of each bed net is the reduction of blood feeding percentage induced by the net when compared to control. The protective effect of each bed net can be calculated as follows:Personal protection (%) = 100 × (Bu − Bt)/Bu, where Bu is the total number of blood-fed mosquitoes in the huts with untreated nets and Bt is the total number of blood-fed mosquitoes in the huts with treated nets .Immediate and delay mortality: the proportion of mosquitoes entering the hut that are found dead in the morning  or after being caught alive and held for 24 h with access to sugar solution  . In thisThe performance of the bed nets were expressed relative to control (untreated nets) in term of:The National Ethics Committee for Health Research of Cameroon approved the protocol of the study (ID:2016/03/725/CE/CNERSH/SP). Written, informed and signed consent was obtained from sleepers before starting the trials. The consent form provided all the information and the evaluation process about the study. Information was translated in local language when needed. All the volunteers involved in the study were followed-up and treated when showing malaria symptoms. All methods were performed in accordance with the relevant guidelines and regulations.GSTe2 mutation was genotyped in each group using an Allele Specific-PCR. This allows a direct measure of the relative survival and feeding success of resistant and susceptible insects in the presence of the different bed nets.The samples collected during the investigation on the performance of nets were grouped in several categories: dead, alive, blood fed, unfed; room and veranda and inside nets. The L119F-GSTe2 mutation was genotyped to assess how the glutathione S-transferase gene, GSTe2, impacts the performance of the bed nets. An allele specific PCR [GSTe2 mutation . The PCR was carried out using 10 mM of each primer and 1 µL of gDNA as template in 15 µL reaction containing 10× Kapa Taq buffer A, 0.2 mM dNTPs, 1.5 mM MgCl2, 1 U Kapa Taq . Amplification was carried out using thermocyclic parameters: 95 °C for 5 min; 30 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 45 s, and final extension at 72 °C for 10 min. The following primers were used: L119F-Fwd: ATG ACC AAG CTA GTT CTG TAC ACG CT; L119F-Rev: TTC CTC CTT TTT ACG ATT TCG AAC T; L119F-Res1: CGG GAA TGT CCG ATT TTC CGT AGA AtAA; L119-F-Sus1: CAT TTC TTA TTC TCA TTT ACA GGA GCG TAaTC. PCR products were separated on 2% agarose gel by electrophoresis. The bands corresponding to different genotypes were interpreted as previously described [Samples classified as dead, alive, blood fed, unfed, room and veranda were used for DNA extraction using the Livak protocol . The L11ific PCR ,28 was uescribed ,28.To calculate the proportion of each entomological outcomes and the level of significance between the treatments and between the control for each entomological outcomes, the XLSTAT software  was used, as done previously ,30. The To investigate the association between the GSTe2 mutation and the ability of the mosquitoes to survive, blood feed or escape, Vassar stats was used to estimate the Odds ratio (OR) based on a fisher exact probability test with a 2 × 2 contingency table.The one-way analysis of variance (ANOVA) using Prism 7.0  was used to determine whether there were any statistically significant differences between the means of the mosquitoes collected from the six huts. In this study, for all the analyses, an alpha of 0.05 was used as the cut off for significance.An. coluzzii susceptible lab strain Ngousso using WHO cone assays. Olyset plus and PermaNet 3.0 showed a mortality of 100 ± 00%, PermaNet 2.0 showed 93.3 ± 3.33% mortality and Olyset gave mortality rate of 96.97 ± 3.33% whereas no mortality was recorded for the untreated net  between the numbers of mosquitoes collected in the different huts was observed  were An. funestus males, 1004 (22%) were Mansonia spp., 03 (0.06%) were An. gambiae ss., 66 (1%) were Culex sp. and 04 (0.08%) were Aedes sp.  were edes sp. A. Howeveanalysis .In comparison to control, the entry rate was significantly reduced in all the huts for the five LLINs with the deterrence rates ranging from 39.2% for PermaNet 2.0 to 60.8% for PermaNet 3.0. PermaNet 3.0 had a significantly higher deterrence compared to PermaNet 2.0 but this was not the case for Olyset Plus (49%) over Olyset (54.9%) .An. funestus s.s. (6.9%). However, the exophily rate was significantly higher in the hut with Olyset , Olyset Plus , PermaNet 2.0  and PermaNet 3.0  compared to the control hut (p < 0.05) (A low exophily rate was recorded in the control hut for trol hut . No sign < 0.05) .An. funestus s.s. (5.4%). However, the mortality rate was significantly higher in the hut with Olyset Plus , PermaNet 2.0  and PermaNet 3.0  compared to the control hut. The mortality rate was significantly higher with the two PBO-based nets than all the pyrethroid-only nets (p < 0.001). However, no significant variation was observed between the mortality in the huts with Olyset  compared to control (p ˃ 0.05), by the huts (p ˃ 0.05) and by the weeks (p ˃ 0.05)  .p < 0.001), Olyset Plus , PermaNet 2.0  and PermaNet 3.0  compared to the control hut (p < 0.05) (High blood feeding rates were recorded in the control hut (40.8%). However, the blood feeding rate (BFR) was significantly lower in the hut with Olyset  .p ˃ 0.05). No significant difference was observed when comparing the PP provided by Olyset and Olyset Plus  (The lowest performance in terms of PP was recorded with PermaNet 2.0 (70.44%). PermaNet 3.0 provided a higher PP when compared to PermaNet 2.0 but not significant  .An. funestus, PermaNet 2.0 had a higher deterrence rate (58.5%) compared to PermaNet 3.0 (36.8%) and similar for Olyset (58.8%) compared to Olyset Plus (52.3%)  .Mansonia spp. (42.4%) than An. funestus (6.9%). However, the exophily rate of Mansonia spp., was significantly lower in the hut with Olyset , Olyset Plus  and PermaNet 3.0  compared to the control hut. For PermaNet 2.0 no significant difference was observed when compared to control  (A higher exophily rate was recorded in the control hut for  ˃ 0.05) .Mansonia spp. was 37.4%. However, this rate was significantly higher in the hut with Olyset Plus , PermaNet 2.0  and PermaNet 3.0  compared to the control. No significant variation was observed between the mortality in the huts with Olyset  compared to control , Olyset Plus , PermaNet 2.0  and PermaNet 3.0  compared to the control hut. PermaNet 3.0 had higher BFI than all the other nets but this was not significant when compared to PermaNet 2.0 and Olyset Plus.The blood feeding rate recorded in the control hut for p ˃ 0.05) but higher than the Olyset nets. No significant difference was observed when comparing the PP provided by Olyset and Olyset Plus  (The lowest PP was recorded with Olyset Plus (67.96%) whereas PermaNet 3.0 provided a higher PP but not significant when compared to PermaNet 2.0  .GSTe2 mutation and the ability to survive exposure to Olyset Plus  when comparing the allelic frequency . A similar result was observed for the genotypic frequencies including between RR vs. SS , RS vs. SS  and RR vs. RS  (Due to the low number of dead mosquitoes (5) the impact on mortality could not be assessed for Olyset net. No correlation was observed between the L119F-21–1.68) .p ˃ 0.05; CI 0.6–2.2)   when comparing the allelic frequency   as well 0.6–6.6) .p ˃ 0.05; CI 0.74–2.7)    but comp 1–11.9) .GSTe2 resistance allele and an increased ability to blood feed (21 blood fed and 92 unfed) when comparing the allelic frequencies  and the genotypic frequency of RS v SS   .p < 0.001; CI 2.26–9.2). An even stronger association was observed when comparing the genotype frequencies between RR vs. SS  and RS vs. SS . But no association was established when comparing RR v RS   when genotyping all the mosquitoes fed and unfed collected in in the hut treated with Olyset (33 blood fed and 103 unfed).For Olyset Plus net, when considering the blood feed and unfed samples from the room only (23 blood fed and 49 unfed) a significant association was found between L119F-GSTe2 and an increased ability to blood feed when comparing the allelic frequencies  . No assop ˃ 0.05; CI 0.55–2.01) .For PermaNet 2.0, no association was observed when comparing the allelic frequency R vs. S  . But an p < 0.05; CI 0.17–0.73) and the genotypic frequency of RS vs. SS   .GSTe2 mutation and exophily (67 in room and 48 in veranda) when comparing the allelic frequency     and the 0.6–2.1) .p < 0.001; CI 1.67–6.9) , RS vs. SS . But no significant association was observed between RR vs. RS  . A stronger association was observed when assessing the impact only among the unfed mosquitoes for RS vs. SS  but not at the allelic level  , RS vs. SS  RR vs. RS   . An even8–18.59) . For Peric level . For Per0.5–1.8) . The sam0.6–4.7) .GSTe2 on the ability of mosquitoes to survive exposure to various nets was assessed using samples from cone assays. Only a few dead were obtained for Yorkool and Olyset, preventing the assessment of these nets.Due to the low number of dead mosquitoes obtained from the experimental huts, the impact of the L119F-p = 1; CI 0.5–1.8) and genotypic for RR vs. SS   at both allelic level  .GSTe2 mosquitoes exhibited a greater ability to survive than susceptible mosquitoes  at both allelic level  and genotypic frequency RR v SS  (Resistant 119F-1.1–4.2) .GSTe2 mosquitoes when exposed to PermaNet 3.0 showed a greater ability to survive than susceptible mosquitoes even than with PermaNet 2.0 at allelic level . A higher correlation was further observed when comparing the genotypic frequencies for RR vs. SS , RS vs. SS  and RR vs. RS  (Resistant 119F-1.8–7.7) . This reGSTe2 on LLINs, we established the association between this marker and the ability to survive exposure to the pyrethroids used for Olyset (permethrin) and PermaNet (deltamethrin) nets besides DDT. Correlation analysis between L119F genotypes and deltamethrin revealed that homozygote resistant mosquitoes were significantly more likely to survive exposure to deltamethrin than both homozygote susceptible SS  and heterozygotes . However, there was no significant difference between RS and SS (p < 0.001) and also more than RS . Contrary to both pyrethroids heterozygote RS mosquitoes are also more able to survive exposure to DDT than homozygote susceptible   .An. funestus and the recent availability of a simple DNA-based diagnostic tool for GSTe2, to establish the impact of GST-mediated metabolic resistance on the effectiveness of both pyrethroid-only and PBO-based LLINs against An. funestus.This study has taken advantage of the predominant presence of metabolic resistance in An. gambiae [An. gambiae in Ivory Coast (Yaokoffikro) for PermaNet 3.0 (54%) and PermaNet 2.0 (34.7%) [An. funestus population in Mibellon, which could potentially be associated with differences in resistance mechanisms. The low level of mortality observed is likely due to the level of pyrethroid resistance in An. funestus in Mibellon with permethrin mortality around 48.88 ± 5.76% and deltamethrin mortality around 38.34 ± 5.79% [An. funestus and Mansonia spp. This higher mortality of PermaNet 3.0 and Olyset Plus with An. funestus is also in line with the cone assay results although PermaNet 3.0 (Top) presented a higher mortality for both huts and cone assays than Olyset Plus as also seen for An. gambiae in Burkina Faso [Mansonia spp.The mortality rates obtained in this study with experimental huts were generally low (7.3 to 30.1%), but such mortality levels are similar to those observed in other experimental hut studies including in Benin , or rece gambiae . However (34.7%)  than see ± 5.79% . Neverthina Faso . Overallp < 0.001) compared with the untreated control (40.8%). However, the blood feeding inhibition rate was higher for PermaNet 3.0 (61.5%) than PermaNet 2.0 (51.4%) this was not the case between Olyset Plus (55.6%) and Olyset (62.4%). Similar results have been obtained for An. gambiae in Benin [An. funestus and Mansonia spp. with PermaNet 3.0 providing the highest inhibition level in Mansonia spp. whereas Olyset Plus was even lower than Olyset.The blood feeding rate of all the five nets, an indicator of the personal protection, provided by the net was significantly lower  was observed for both An. gambiae and Mansonia spp. in experimental huts [An. funestus, this was not the case in Mansonia spp. with no significant change observed probably as Mansonia spp. are more exophilic [The excito-repellency effect of all the nets was at least twice higher compared to control showing that squitoes ,32. No sxophilic .-GSTe2 diagnostic tool [With the design of the L119Ftic tool , this stAn. funestus and the L119F-GSTe2 mutation as mosquitoes with the 119F resistance allele have significantly higher blood feeding rate compared to those with L119 susceptible allele against Olyset Net (p < 0.001) and Olyset plus (p < 0.001). This is similar with observations for the cytochrome P450s CYP6P9a and CYP6P9b for which the resistant alleles CYP6P9a_R and CYP6P9b_R were recently shown, in a release-recapture experimental hut study, to also provide a greater ability to blood feed and to survive exposure to pyrethroid-only LLIN (PermaNet 2.0) [GSTe2 mutation likely contributes to an increased malaria transmission as every additional bite increases the chance of sporozoite to be passed to the populations. This is particularly a concern as 119F-RR resistant mosquitoes have also been shown to live longer [Plasmodium [-GSTe2 on blood feeding ability is mainly seen for Olyset Plus and Olyset LLINs impregnated with permethrin but present an opposite effect for PermaNet 3.0 and 2.0 both impregnated with deltamethrin. The cause of such difference between permethrin- and deltamethrin-based nets is unclear at this point since the greater blood feeding in Olyset net is not associated with greater survivorship. Functional analyses with transgenic Drosophila, In vitro metabolism assays with recombinant GSTe2 enzyme combined with genotype/phenotypes analyses had shown that GSTe2 was able to confer resistance to both permethrin and deltamethrin but more so to permethrin [kdr) on resistance did not reveal such a significant association with blood feeding potentially due to the fact that kdr frequency was already very high in tested An. gambiae population [kdr effect in two separate populations of An. gambiae in Benin; one with high kdr frequency (Ladji) and another with low frequency  did suggest that kdr could also impact the efficacy of LLINs [A significant association was observed between the blood feeding ability of Net 2.0) ,34. Thise longer ,28 and tasmodium . Howeverrmethrin ,36. Thispulation . HoweverCulex pipiens [Interestingly, the L119F-GSTe2 mutation was also associated with an increased exophily for Olyset Plus and PermaNet 2.0 suggesting that the increased expression of GSTe2 could impact mosquito’s behavior helping them avoids exposure to insecticides. Similarly, over-expression of carboxylesterase in  pipiens  has been pipiens  supportiGSTe2 and mortality in the experimental hut’s samples for all LLINs potentially due to the low number of dead mosquitoes which did not provide statistical power coupled with lower frequency of RR in this location. However, a trend was observed for PermaNet 3.0 with an increased proportion of 119F homozygote resistant mosquitoes able to survive exposure to this LLIN than both homozygote susceptible L119-SS  and heterozygotes .No significant association was observed between L119F-However, other mechanisms than GSTs are also at play in this location of Mibellon, as shown recently , revealiGSTe2 and reduced mortality with LLINs was supported by results from cone assays where a very strong association was observed between L119F-GSTe2 and ability to survive exposure to PermaNet 3.0 (side) (OR = 30.1) and PermaNet 2.0 although at lower extent (OR = 2.09). The lack of association observed with Olyset Plus could be explained by the effect of PBO which for this net is incorporated on the entire net whereas for PermaNet 3.0 PBO is only on the top. The PermaNet 3.0 (side) used here does not contain PBO. One reason why a greater association is found with PermaNet 3.0 (side) could be the fact that with higher deltamethrin concentration than PermaNet 2.0, mosquitoes surviving exposure to PermaNet 3.0 are those likely to be much more resistant because of a greater expression of GSTe2 and other cytochrome P450s. So the higher dose of deltamethrin in PermaNet 3.0 has further selected mosquitoes with 119F-GSTe2 resistant allele than with the lower dose in PermaNet 2.0. This is also the likely reason why a trend of a greater ability to survive was seen in the experimental hut for PermaNet 3.0.This suggests that mosquitoes displaying several resistance mechanisms than just P450-based resistance could avoid even PBO-based nets as when cytochrome P450 enzymes are inhibited by PBO, mosquitoes bearing other mechanisms, as in this case the 119F resistance allele, are able to better survive exposure to PBO-based nets. However, this cannot explain why such results were not observed with Olyset Plus the other PBO-net. However, the spread of a GSTe2-mediated metabolic resistance conferring the ability of mosquitoes to survive exposure to synergist LLINs will constitute a concern at a time when such nets are gradually being introduced to fight pyrethroid resistant mosquitoes. GST resistance will also need to be taken into consideration than just cytochrome P450 resistance. Nevertheless the greater efficacy seen with the roof of PermaNet 3.0 in cone bioassays shows that P450 role remains important. Future studies will need to also perform synergist assays with DEM and PBO to establish the contribution of both GSTs and cytochrome P450s to better inform of the likely efficacy of LLINs.The trend for a correlation between An. funestus in Cameroon using experimental huts. However, PBO based nets (PermaNet 3.0 and Olyset Plus) are more efficient than conventional pyrethroid-only nets. It is the reason why as suggested by WHO 2017 report we recommend the deployment of PBO nets in area where pyrethroid resistance is confirmed and at least partly conferred by P450. Noticeably, the GST-mediated metabolic resistance not impacted by PBO could threaten the continued effectiveness of the PBO-based LLINs and the impact of this mechanism needs also to be considered to maximize the effectiveness of LLINs. One option could be to generate new mosaic LLINs also incorporating the Diethyl Maleate (DEM) beside PBO to inhibit GST metabolic activity and further increase the efficacy of these nets. However, further studies will need to be performed before including establishing the extent of synergism provided by DEM in various mosquito populations and the most suitable position on the net (either on top as PBO or on the entire net).This study has revealed a loss of efficacy of LLINs against pyrethroid resistant populations of


The inclusion of crude glycerin at 21% in to total mixed ration (TMR) diets with chitosan supplementation at 2% enhanced ruminal propionate concentration and reduced methane production without causing any detrimental effect on the gas kinetics or nutrient digestibility.Methane  and chitosan levels were added at 0, 1, and 2% dry matter (DM) of substrate. Then, 0.5 g of TMR substrates were added into 40 mL bottles, together with respective doses of chitosan and then incubated at 39 °C. The dietary treatments were performed in three replicates within the incubation, and incubations were repeated on three separate days (runs). No interactions were found between crude glycerin and chitosan doses in terms of theoretical maximum of asymptotic gas production (b), rate of gas production (c), the discrete lag time prior to gas production (L), or the cumulative gas production at 96 h of incubation (p > 0.05). Cumulative gas production at 96 h of incubation was similar among the doses of crude glycerin and levels of chitosan, which ranged from 64.27 to 69.66 mL/g DM basis of substrate (p > 0.05). The concentration of ruminal NH3-N after 2 and 4 h of incubation ranged from 14.61 to 17.10 mg/dL and did not change with the addition of crude glycerin with chitosan (p > 0.05). The concentration of CH4 after 2 h of incubation did not change among treatments (p > 0.05), whereas after 4 h of incubation, CH4 synthesis was significantly reduced by enhancing doses of crude glycerin and chitosan (p < 0.05). The combination of 21% of crude glycerin in TMR with 2% chitosan depressed CH4 production as much as 53.67% when compared to the non-supplemented group. No significant crude glycerin and chitosan interaction effect was detected for in vitro digestibility of nutrients after incubation for 12 and 24 h using the in vitro gas production technique (p > 0.05). In addition, no significant changes (p > 0.05) were observed in total volatile fatty acids, acetate (C2) or butyrate content among treatments and between the main effects of crude glycerin with chitosan. At 4 h of incubation, ruminal C3 content and the C2 to C3 ratio changed significantly when crude glycerin and chitosan was added (p < 0.05). The 21% crude glycerin incorporate into TMR, in combination with 2% additional chitosan, increased C3 content by 26.41%, whereas the ratio of C2 to C3 was reduced by 31% when compared to the control group. Propionate concentration increased by 11.75% when increasing levels of chitosan at 2% of substrate, whereas the C2 to C3 ratio decreased by 13.99% compared to the 0% chitosan group. The inclusion of crude glycerin at 21% in TMR diets with chitosan supplementation at 2% enhanced ruminal propionate concentration and reduced methane production without causing any detrimental effect on the gas kinetics or nutrient digestibility.It was hypothesized that the combination of glycerin and chitosan improves ruminal fermentation efficiency via an enhanced propionate (C3) and reduces in vitro CH Methanese gases .4 production. Lee et al. [4 formation in alfalfa and corn by 25.2% and 14.8%, respectively, thus reducing the methanogenic potential of forages, so the addition of glycerin might be an influential means of inhibiting ruminal CH4 formation. Similarly, van Cleef et al. [4 synthesis and negatively affecting microbial synthesis and feed digestion. However, the studied results differed in that they indicated no change in CH4 production when supplemented with glycerin [4 production results, such as the levels used, the purity of the glycerin and the in vitro substrate diets. Therefore, an evaluation of glycerin is still necessary to elucidate the specific factors to confirm their effects on ruminal CH4 production.Crude glycerin is a residue from biodiesel production . In the e et al.  indicatef et al.  indicateglycerin . This co4 production [4 reduction could be due to the degree of deacetylation found in the chitosan, which may modify the permeability of methanogenic bacteria cell walls [4 synthesis when high concentrations are added to the substrate. Furthermore, adding chitosan at 2% of substrate resulted in a strong reduction effect on CH4 formation [Chitosan is a biopolymer found in the exoskeletons of crab and shrimp. Interestingly, chitosan can shift volatile fatty acid (VFA) profiles and change ruminal fermentation, as well as increase C3 concentration and, consequently, reduce CHoduction . Furtherll walls . A previll walls  demonstrormation .4 production. This was explored through in vitro gas production with substrates containing crude glycerin, which replaced cassava chips in the studied ration.Therefore, it was hypothesized that the combination of glycerin and chitosan improves ruminal fermentation efficiency via an enhanced C3 and reduces in vitro CHAnimals procedures were allowed by the Animal Ethics Committee of Khon Kaen University (protocol number 9/2561), based on the Ethic of Animal Experimentation of National Research Council of Thailand.w/v) at 26–30 °C for 16 h. The residue was rinsed and soaked in tap water for 6–8 h. It was then removed from the water, and protein was removed using 0.62 M NaOH solution (1:5 w/v) at 26–30 °C for 20 h. The chitin product was stripped of acetyl groups using NaOH at 65 °C for 20 h, after which the chitosan was obtained. The chitosan was rinsed and dried in sunlight before being used for chemical composition analysis and further investigation.Chitosan in the experiment was prepared using Toan’s method . BrieflyCrude glycerin used in the experiment was offered from the Specialized Research and Development Center for Alternative Energy from Palm, Prince of Songkla University, Thailand and chemical contents are presented in In experiments, two male, rumen-fistulated dairy cattle weighing 370 ± 50 kg of body weight were supplied as rumen fluid donnors. The animals were fed with TMR1 , volatile fatty acids (VFAs) and CH4 determination. A digestibility evaluation was arranged with different set of 54 bottles (3 bottles per treatment × 9 treatments × 2 sampling times at 12 and 24 h incubation).The experimental design was organized following a 3 × 3 factorial in completely randomized design and treatments are the combination of the 3 levels of glycerin   and 3 leChemical analysis was performed on the TMR and chitosan. Dry matter (DM), ash, crude protein (CP), organic matter (OM), ether extract (EE), and acid detergent fiber (ADF) were measured according to the Association of Official Analytical Chemists (AOAC) . The neug for 15 min, and the supernatant was analyzed for NH3-N by Kjeltech Auto 1030 analyzer . Total VFA, C2, C3, and C4 were evaluated using high-performance liquid chromatography (HPLC) . In vitro digestibility was elucidated at 12 and 24 h of incubation, when the content was filtered through pre-weighed Gooch crucibles (40 mm of porosity) and residual DM was calculated. The difference in weight of DM and its components before and after incubation represented in vitro DM digestibility (IVDMD) and in vitro organic digestibility (IVOMD), in vitro NDF digestibility (IVNDF), and in vitro ADF digestibility (IVADF) [4 using gas chromatography .After incubation for 0, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 48, 72, and 96 h, gas production was measured by using a pressure transducer and a calibrated syringe. Fermentation liquor was collected at 2 and 4 h of incubation to investigate the ruminal pH and then filtered through 4 layers of cheesecloth. The fermentation liquor was centrifuged at 16,000 × b is the asymptotic gas production (mL/1 g DM); c is the rate of gas production (/h) and L (h) is the discrete lag time prior gas production.Estimates of the kinetic parameters of gas production (mL/g DM basis) were obtained using the NLIN procedure according to France et al.  using th4, NH3-N, pH, VFAs and digestibilities were subjected to the general linear model (GLM) procedures of SAS [Data of in vitro gas kinetics, cumulative gas production, CHs of SAS . DiffereThe experimental diet’s formulation and nutritional content are shown in The removal of protein and minerals from shrimp shell, or chitin, was determined for ash and CP content to evaluate whether these critical values are lower than 1% . The chib), the rate of gas production (c), the discrete lag time prior to gas production (L), or the cumulative gas production at 96 h of incubation  and ranged from 66.86 to 72.25 mL/g DM basis. Cumulative gas production at 96 h of incubation was similar among the doses of crude glycerin and levels of chitosan, which ranged from 64.27 to 69.66 mL/g DM basis of substrate (p > 0.05). These results demonstrated that increasing doses of crude glycerin to 21% and chitosan to 2% did not adversely influence gas kinetics and cumulative gas production. This is in agreement with Avila et al. [No interactions were found between crude glycerin and chitosan doses in terms of theoretical maximum of asymptotic gas production  and demonstrated a consistent range of 6.65 to 6.76. Considering it is a buffered medium which is an optimum pH for ruminal maintenance and microbial activity. The concentration of ruminal NH3-N after 2 and 4 h of incubation ranged from 14.61 to 17.10 mg/dL and did not change with the addition of crude glycerin with chitosan (p > 0.05). This could be due to the TMR diet, which was isonitrogenous and the crude glycerin with chitosan introduced no N source to the incubation (4 after 2 h of incubation was not different among treatments (p > 0.05) which might be due to the initial ruminal fermentation process, thus lowering substrate for CH4 to synthesis. However, after 4 h of incubation, CH4 synthesis was significantly reduced by enhancing doses of crude glycerin and chitosan (p < 0.05). The influence of crude glycerin with chitosan on pH, NHcubation . The con4 synthesis, Vito et al. [4 could be reduced with glycerin addition because of the adverse influence of glycerin on methanogen populatios. Vito et al. [4 production. Furthermore, Vito et al. [Entodinium and Isotricha when the increasing level of crude glycerin could affect CH4 reduction. In addition, Lee et al. [4 synthesis in the rumen, suggesting an advantageous impact of glycerin on enhancing energy utilization. The small CH4 production in the present work might be ascribable in part to the small NDF concentration of the TMR and the use of crude glycerin, which replaced cassava chips [4 production, and TMR3 produced 22.03 mL/1 g less CH4 compared with TMR1, which produced CH4 at 36.51 mL/1 g DM basis.Methane concentration was inhibited by 39.65% when 21% crude glycerin was included in TMR, compared to the TMR group with 0% crude glycerin. Regarding the influence of crude glycerin on in vitro CHva chips . Higher 4 production by 26.05% compared to no chitosan supplementation. Similarly, a substantial decrease in CH4 production (~42%) was found when highly soluble chitosan (98.70% deacetylated) was added at 7% to a diet by Rusitec systems [4 synthesis without an adverse effect on diet digestion could be accomplished by reducing chitosan solubility (88.00% deacetylated). This agrees with previous reports which indicated no adverse effect of chitosan on nutrient digestibility when little roughage was used in vivo [4 production decreased when chitosan was added at 2% of substrate. The mode of action by which chitosan reduces CH4 synthesis in ruminants has been proposed. This could be due to properties such as chitosan’s high degree of deacetylation, which may modify methanogen’s cell wall permeability, thus reducing CH4 production [Supplementation of chitosan at 2% could reduce CH systems . The pre in vivo ,23 or inoduction  proposed4 production as much as 53.67% when compared to the nonsupplemented group. Interestingly, it has been reported that crude glycerin and chitosan could shift the VFA profile and change ruminal fermentation, as well as increase C3 concentration, resulting reduced CH4 production [2 sink and could support in the conversion of carbohydrate fermentation from C2 to C3 production. The conversion could affect the overall electron equilibrium in the rumen and decrease the availability of hydrogen for CH4 synthesis. This result could confirm that the combination of crude glycerin and chitosan might reduce CH4 production more effectively than individual supplementation. However, the present study found CH4 was reduced in the early fermentation of 4 h of incubation, thus the inhibitory effect of glycerin and chitosan throughout the complete fermentation process (96 h of incubation) should be further elucidated.The combination of 21% of crude glycerin in TMR with 2% chitosan depressed CHp > 0.05). Increasing levels of crude glycerin up to 21% DM in TMR diet did not change IVDMD, IVOMD, IVNDFD, or IVADFD after 12 and 24 h of incubation (p > 0.05). In vitro dry matter digestibility at 12 and 24 h of incubation ranged from 53.34% to 56.29% DM and 65.70% to 67.39% DM, respectively, for all levels of crude glycerin inclusion. This result indicated that crude glycerin could be a substitute energy source in TMR diets without adversely affecting feed utilization and agreed with Schröder and Südekum [No significant crude glycerin and chitosan interaction effect was detected for in vitro digestibility of nutrients after incubation for 12 and 24 h using the in vitro gas production technique . At 24 h of incubation, the IVNDFD and IVADFD when adding chitosan ranged from 62.33 to 64.75% DM and 50.88 to 53.83% DM, respectively. The chitosan shifted fermentation toward a pathway with greater energetic potential without adversely affecting feed digestion. This result agreed with previous studies. Goiri et al. [Supplementation of chitosan levels did not significantly alter the in vitro digestibility of nutrients . In addition, no significant changes (p > 0.05) were observed in TVFA, C2, or C4 content among treatments and between the main effects of crude glycerin with chitosan. At 4 h of incubation, ruminal C3 content and the C2 to C3 ratio changed significantly when crude glycerin and chitosan was added (p < 0.05). The 21% crude glycerin incorporated into TMR, in combination with 2% additional chitosan, increased C3 content by 26.41%, whereas the ratio of C2 to C3 was reduced by 31% when compared to the control group. The sample’s C3 proportion increased with higher amounts of crude glycerin in the TMR, this increase being mainly at the expense of C2. The main effect of crude glycerin inclusion (21% vs. 0% in TMR) was an 18.69% increase in C3 content and a 22.35% reduction in the C2 to C3 ratio. This likely occurred because crude glycerin, which is similar to a fermentable carbohydrate source, underwent ruminal fermentation to C3. Van Cleef et al. [Selenomonas bacteria and mostly used by ruminants during the first 4–6 h after digestion. Almeida et al. [4 emissions, as shown in There were no interactions on total TVFA and VFA profiles between crude glycerin and chitosan (a et al.  found thThe enhancement in the C3 concentration and the decrease in the C2 to C3 ratio in ruminal fluid are in agreement with previous studies. Avila et al.  found thPropionate concentration increased by 11.75% when increasing levels of chitosan at 2% of substrate, whereas the C2 to C3 ratio depressed by 13.99% compared to the 0% chitosan group. Zanferari et al.  found thBased on the present in vitro study, the inclusion of crude glycerin at 21% in TMR diets with chitosan supplementation at 2% enhanced ruminal propionate concentration and reduced methane production by 54% without causing any detrimental effect on gas kinetics or nutrient digestibility. However, an additional further study on a population of methanogen bacteria influenced by crude glycerin and chitosan is needed.


The aim was to assess growth velocity (GV) during human recombinant growth hormone (hGH) treatment of children with multiple pituitary hormone deficiency (MPHD) caused by pituitary stalk interruption syndrome (PSIS) and to analyze the characteristics of patients that attained normal adult heights.Data from 74  children with MPHD caused by PSIS with GH, thyroid stimulating hormone, gonadotropin and adrenocorticotropic hormone deficiencies were collected. Subjects were divided into groups: 12 pre-pubescent females  and 36 pre-pubescent males . The remaining 22 males were further sub-divided into two groups  according to the initiation of gonadotropin replacement treatment, based on bone age and height.No differences in change in height standard deviation score (△HtSDS) and GV were observed at different time points of hGH treatment between the Female-Group and Male-Group 1 (p>0.05). GV was significantly greater in the first year of hGH therapy than in subsequent years: Female-Group p=0.011; Male-Group 1 p<0.001; Male-Group 2 p=0.005; and Male-Group 3 p=0.046. Adult height was achieved by 23  patients. The total gain in height positively correlated with the GV during the first year .GV during hGH treatment were similar amongst pre-pubescent males and females with MPHD caused by PSIS. GV during the first year of hGH treatment appears to be an effective predictor of final height in patients with MPHD caused by PSIS. Pituitary stalk interruption syndrome (PSIS) is a kind of congenital disease associated with multiple pituitary hormone deficiency (MPHD). Human recombinant growth hormone (hGH) treatment is the optimal therapy for short stature in children with isolated GH deficiency and can effectively increase growth velocity (GV) to attain adult heights within the target range.GVs during hGH treatment were similar amongst pre-pubescent males and females with MPHD caused by PSIS. The GV during the first year of hGH treatment is an effective predictor of future height outcomes in patients with MPHD caused by PSIS.Pituitary stalk interruption syndrome (PSIS) is characterized by the occurrence of a thin or absent pituitary stalk, hypoplasia of adenohypophysis, and ectopic neurohypophysis on magnetic resonance imaging (MRI) of the hypothalamo-pituitary region . It is aPSIS as the most common cause of MPHD, was first reported by Fujisawa et al  in 1987.In this study, we assessed the growth velocity (GV) of patients with MPHD caused by PSIS who were administered hGH. We further analyzed the characteristics of patients that subsequently attained adult heights.via gonadotropin or sex hormone treatment. Pre-pubescent subjects at presentation were divided into the Female-Group and Male-Group 1. Additionally, patients were treated with hGH followed by gonadotropin, administered at the appropriate age and the male patients from this subgroup formed Male-Group 2. The final study group consisted of male patients who received initial hGH and gonadotropin treatment simultaneously and these were designated Male-Group 3.In this retrospective study, data from patients diagnosed with PSIS during childhood and adolescence presenting during the time period from January 2008 to November 2018 in pediatric endocrine outpatients of Shandong Provincial Hospital were analyzed. Patients with confirmed GH, thyroid stimulating hormone (TSH), gonadotropin and adrenocorticotropic hormone (ACTH) deficiencies were included in the analysis. None of the patients had spontaneous puberty and puberty was induced The study was approved by the Medical Ethics Committee of Shandong Provincial Hospital, affiliated to Shandong University . Patients or their parents/guardians provided verbal consent for anonymized data to be collated and analyzed, approved by the ethics committee.The diagnosis of PSIS was based on cranial MRI of the hypothalamus and pituitary gland. Imaging criteria for a diagnosis of PSIS included an absent or thin pituitary stalk, hypoplasia of the anterior pituitary gland, and ectopic location of the posterior pituitary. MRI scans were performed using a 3.0 T Scanner  in the sagittal and coronal planes on T1 and T2 weighted imaging (slice thickness 3 millimeters).Bone age (BA) was determined by left hand and wrist X-ray images according to the method of Greulich and Pyle. Hypophyseal hormone levels were investigated in each patient. The pituitary axis was examined using the following tests:  GH deficHeight was measured in centimeters (cm) in the morning by the same medical team. Height measurements were standardized for age and sex and expressed as standard deviation scores (HtSDS) relative to chronological age (CA) according to Growth Charts for Chinese Children and Adolescents (2009) ,11. The Weight was measured in kilogrammes (kg) in the morning, in the fasting state with no shoes and light clothes at each visit. Body mass index (BMI) was calculated using the standard formula of weight (kg) divided by height squared (meters). BMI values were transformed into BMI-SDS, based on Normative Values for Chinese Children and Adolescents (2009) ,11 to ad2/day, oral administration, divided into two daily doses. The dosages of L-thyroxine were between 1.5-2.0 ug/kg/day, oral administration, qd. The dosages of hydrocortisone and L-thyroxine were adjusted to maintain the levels of fT4, COR, blood glucose and serum electrolytes within normal ranges. In patients with normal thyroid and adrenal hormone levels (including patients who were corrected with hydrocortisone and L-thyroxine) hGH was administered . The dosage of hGH was between 0.10-0.15 IU/kg/day) and administered by daily injection 7 days/week. Puberty was initiated in 26 patients through the administration of gonadotropin; exogenous human chorionic gonadotropin (hCG) and/or urine-derived human menopause gonadotropin (hMG) therapy . For male patients the dosage of intramuscular (i.m.) hCG was 2000 IU, twice per week, whilst for female patients 75 IU i.m. hMG was administered twice a week. Boys had a pretreatment phase of hCG for three months. As serum testosterone (TO) reached normal values, hCG combined with hMG was administered to improve sexual development. If the TO concentration did not attain normal values, exogenous TO  was given to induce puberty . The continuous data in our research were tested for normal distribution using Kolmogorov-Smirnov test, and found to be approximately normally distributed. The descriptive statistics of the quantitative variables were presented as means ± standard deviations (SD). Groups were compared using the Student’s t-test. Pearson’s correlation was used to assess the relationships between various parameters. The threshold for statistical significance was <0.05.In total, data from 74 patients  was analyzed. Amongst these patients, 48  who received hGH treatment and other deficient hormones, excluding gonadotropin during the study period, were pre-pubescent. Pre-pubescent subjects at presentation were divided into the Female-Group (n=12) and Male-Group 1 (n=36). Additionally, 13 patients  were treated with hGH followed by gonadotropin which was administered at the appropriate age to induce puberty. The 10 male patients in this latter group formed Male-Group 2. Lastly, 13 subjects  received combined hGH and gonadotropin treatment at presentation. These 12 males were grouped into Male-Group 3.CA, BA, BMI-SDS and HtSDS during hGH treatment were 8.51±3.08 years, 4.91±2.75 years, -0.20±1.11 and -3.60±1.76 in the Female-Group and 8.94±3.42 years, 5.51±3.22 years, 0.20±1.35 and -2.97±1.23 in the Male-Group 1. There were no differences in CA and BA, BMI-SDS and HtSDS at hGH treatments between pre-pubescent females  and males  . The CA was significantly larger than the BA in both groups  .There were no differences in HtSDS at any point during hGH treatment (p=0.292 and p=0.157). For Female-Group patients, the GV was 11.63±2.38 cm/y in the first year and 9.37±1.48 cm/y in the second year. The GV of Male-Group 1 in the first year was 11.95±2.62 cm/y compared to 9.83±1.71 cm/y in the second year. No differences in GV at any time point during hGH treatment between pre-pubescent females  and males  (p=0.710 for the first year and p=0.410 for the second year) were observed. GV in the first year was higher than in the second year for both two groups  . The △HtDetailed information of CA, BA, height SDS and GV during hGH or gonadotropin treatment are shown in The GV during the first two years of hGH treatment in the Male-Group 2 were 13.37±2.45 and 10.08±2.16 cm/year. GV during the first two years of hGH (hGH + gonadotropin) treatment of Male-Group 3 were 10.68±3.59 and 8.16±2.03 cm/year. We observed no differences in GV during the first year of hGH treatment amongst the three groups . During the second year, no differences in GV were observed between Groups 1 and 2 (p=0.701), but the GV of Group 3 was significantly lower than the other two male groups .GV during the first year of hGH treatment was higher than the second year for the three groups  . The difThe CA during the initiation of hGH + gonadotropin treatment of Male-Group 2 (13.39±2.80) were significantly lower than those of Male-Group 3 (p=0.007), but no differences in BA at the initiation of hGH + gonadotropin treatment between the groups were observed (p=0.066). The GV in the first year of hGH + gonadotropin treatment in Group 3 was significantly higher than that of Group 2 (p=0.041), which was similar for △HtSDS (p=0.004).th percentile (adult height ≥172.7 cm or adult HtSDS ≥0). For females, all patients (4/4) reached a normal adult height range and all were above the 50th percentile (adult height ≥160.6 cm or adult HtSDS >0).In total, 23 patients  with PSIS reached adult height following hGH treatment. For male patients, 18/19 attained adult height >-2 SD, yet only seven reached a height above the 50The mean adult height was 168.5±6.1 cm (HtSDS=-0.47±1.11) for males and 164.0±2.9 cm (HtSDS=0.77±0.49) for females. The parental target height was 170.1±4.9 cm (HtSDS=-0.43±0.82) for males and 160.8±1.3 cm (HtSDS=0.26±0.03) for females. The mean age at initiation of hGH treatment in females was 10.4±0.8 years and 14.4±3.5 years in males. The mean BA at initiation of hGH treatment in females was 8.4±0.8 years and 9.9±3.5 years for males. Mean HtSDS at hGH treatment onset was -3.11±1.86 for males and -1.75±0.23 for females, respectively. The mean GV in the first year of hGH treatment were 11.0±3.2 cm and 12.9±1.9 cm for males and females, respectively. The mean total height gain was 23.9±15.6 cm and 20.9±4.9 cm for males and females, respectively.A negative correlation was found between the total height gain and BA at hGH onset and between total height gain and height at hGH treatment onset . Moreover, a positive correlation was observed between total height gain and GV in the first year of hGH treatment . Various anterior pituitary hormone deficiencies and clinical presentations are common in PSIS patients. To date, studies on the growth of children and adolescents with PSIS during the course of hGH treatment are sparse and continuous follow-up to adult age is rarely reported . In thisAll children short in stature can receive hGH treatment to decrease linear height deficits. The CA of PSIS patients receiving hGH treatment in this study were older than those of previous studies . ChineseIn previous studies on congenital IGHD patients, the GV in the first year of hGH therapy was 8.92±2.99 cm for males and 8.17±3.15 cm for females . The GV Although the mean CA and mean BA of Male-Group 3 were both higher than those of the other two male groups, the GV during the first two years of hGH treatment was similar, which may be explained by the induction of puberty. The induction of puberty leads to a growth spurt which may explain the response to hGH treatment observed.In previous GHD studies, only one third of patients reached normal adult height in response to hGH treatment . The durA positive correlation between total height gain and GV in the first year of hGH treatment was in accordance with previous studies . The totThe uneven number of male and female patients and differences in duration of hGH treatment may have influenced the results. Of particular note there were only four females in this study who attained adult height. It is not possible to reach a definite conclusion about the effect of hGH in female PSIS patients and the number of female patients is too low to compare with male patients reliably In addition data regarding the sexual development of PSIS patients was not collected and so analysis of the effect of hGH and gonadotropin therapy on pubertal development in PSIS patients was not performed. These limitations should be addressed in future studies.Males and females with MPHD caused by PSIS had a similar GV during hGH treatment before puberty. The GV during the first year of hGH treatment can predict future height outcomes for patients with MPHD caused by PSIS. PSIS patients may attain normal adult heights following hGH treatment.


The main purpose and research question of the study are to compare the efficacy of high-security closed versus open devices for human oocytes’ vitrification.A prospective randomized study was conducted. A total of 737 patients attending the Infertility and IVF Unit at S.Orsola University Hospital  between October 2015 and April 2020 were randomly assigned to two groups. A total of 368 patients were assigned to group 1 (High-Security Vitrification™ - HSV) and 369 to group 2 (Cryotop® open system). Oocyte survival, fertilization, cleavage, pregnancy, implantation, and miscarriage rate were compared between the two groups.P = .297). A total of 4029 oocytes were vitrified (1980 and 2049 in groups 1 and 2 respectively). A total of 2564 were warmed (1469 and 1095 in groups 1 and 2 respectively). A total of 1386 morphologically eligible oocytes were inseminated by intracytoplasmic sperm injection .No statistically significant differences were observed on survival rate (70.3% vs. 73.3%), fertilization rate (70.8% vs. 74.9%), cleavage rate (90.6% vs. 90.3%), pregnancy/transfer ratio (32.0% vs. 31.8%), implantation rate (19.7% vs. 19.9%), nor miscarriage rates (22.1% vs. 21.5%) between the two groups. Women’s mean age in group 1 (36.18 ± 3.92) and group 2 (35.88 ± 3.88) was not significantly different (The present study shows that the replacement of the open vitrification system by a closed one has no impact on in vitro and in vivo survival, development, pregnancy and implantation rate. Furthermore, to ensure safety, especially during the current COVID-19 pandemic, the use of the closed device eliminates the potential samples’ contamination during vitrification and storage. Oocyte storage is used to preserve fertility for medical or social reasons –5 and toThe principle of oocytes and embryo vitrification is to fully eliminate ice formation in the medium that contains the sample, in phases of cooling, storage, and warming of the procedure . It can The cryoprotectant takes part in lowering the freezing point and in reducing or preventing the formation of ice crystals in aqueous solutions. The addition of cryoprotectants into vitrification solutions results in a significant increase in the degree of cellular dehydration. During the warming procedure, the transfer of the oocytes from a solution containing a high concentration of cryoprotectant to an isotonic solution can also lead to a reverse osmotic shock or over-swelling that could be lethal . TherefoVery high cooling and warming rates, high cryoprotectant concentrations solutions, and a low volume containing the samples are crucial requirements for success human embryos and oocytes’ vitrification –22.Over the years, various authors have described different vitrification protocols. They differ in the type of cryoprotectants used  . To date2) and usually provide very high cooling  rates. Conversely, closed devices . “Open” vitrification devices  are una CryoTip . Some st CryoTip –38. Furt CryoTip –41. Desphe tanks –44. The 196.5 °C , 45. Durfication –48.2 and thus avoids hypothetical contamination particularly dangerous especially during and after the COVID-19 pandemic.The main purpose and research question of the study are to compare the efficacy of high-security closed versus open devices for human oocytes vitrification. In the case of comparable efficacy, closed systems might offer an additional advantage as the samples are not in direct contact with LNThe study was approved by the local ethical committee. All the patients attending the Infertility and IVF Unit, at Sant’Orsola University Hospital , were stimulated with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone analogues. Monitoring was performed with estradiol serum levels’ determinations and pelvic ultrasounds to measure the diameter of the follicles. Ovulation was triggered by an injection of subcutaneous human chorionic gonadotropin (hCG) when the leading follicle reached 18–20 mm diameter and estradiol serum concentrations were around 200 pg/mL for each mature. Vitrification was performed in the occurrence of supernumerary oocytes, failure of semen production, and risk of ovarian hyperstimulation syndrome. For ethical reasons, the Bologna Infertility and IVF Unit cryopreserve female gametes instead of embryos. For this reason, just a few fresh or warmed oocytes are inseminated each time.This prospective randomized study was designed to compare the efficiency of vitrification in open versus closed devices. The primary end-point was the survival rate after warming.α 0.05 and power of 80%), based on the average results previously obtained (data not shown).francesca.labriola2@unibo.itA total of 1377 oocytes were needed in each arm assuming a survival rate of 65% for High-Security Vitrification™ and 75% for Cryotop® . A total of 4029 MII oocytes were vitrified. Oocytes were randomly assigned to the closed group (group 1) or the open group (group 2) , using Kuwayama’s protocol . Oocytes2. In the open system, the straw cap was initially removed under LN2 where the samples were in direct contact, and the carrier quickly submerged in 1 mL preheated TS. Regardless of the system used, the samples remained in the TS solution for 1 min at 37 °C. Then, oocytes were transferred into a drop of dilution solution (DS) at room temperature (25 °C) and incubated for 3 min. After two subsequent washing procedures in washing solution (WS) at room temperature for 6 min in total, oocytes were transferred into the fertilization medium (Vitrolife). Warmed oocytes were considered morphologically surviving if there were no dark/degenerated or contracted ooplasm and no cracked zona pellucida. Subsequently, oocytes were cultured at 37 °C (6% CO2 and 5% O2). Two hours post warming, oocytes were morphologically re-evaluated. Regardless of semen parameters, intracytoplasmic sperm injection (ICSI) was performed only in non-degenerate oocytes and completely rehydrated. The injected oocytes were cultured in individual 50-μL droplets of cleavage medium (Vitrolife) under oil (Vitrolife). The uninseminated oocytes surviving the warming process have been discarded because they are morphologically not eligible. Oocytes are considered morphologically not eligible if they have one or more of these anomalies: dark zona pellucida, large perivitelline space, dark or granular cytoplasm, cytoplasmic inclusions, vacuoles, and shape abnormalities.Thaw Kit® (Irvine Scientific) was used to warm oocytes according to Kuwayama’s protocol . In the Fertilization was assessed 16–20 h post ICSI by an inverted microscope (× 20).Embryo quality was assessed on day 2 using Veeck’s classification .Embryos were transferred on day 2. Endometrial preparation was performed with patches containing estradiol hemihydrate: 300 μg/day for 11 days, and when the endometrial thickness reached 10 mm, 600 mg/day of micronized progesterone was added.Pregnancy was assessed by serum hCG measurement 14–15 days after embryo transfer  and was then confirmed when at least one gestational sac was visualized at the transvaginal US after two further weeks. Miscarriage was considered the loss of a clinical pregnancy before the 20th week of gestation.t test was used to compare aged, frozen, and warmed oocytes. The chi-square test was used for oocyte’s survival, fertilization, cleavage, number of embryos transferred, pregnancy, implantation, and miscarriage rate. P values < .05 were considered statistically significant. All tests performed were validated through IBM SPSS Statistics (version 25.0).Data were expressed as mean ± SD (standard deviation). Student’s Tables A total of 737 patients for a total of 775 vitrification cycles were analyzed in the present study: 368 (389 vitrification cycles) and 369 (386 vitrification cycles) in groups 1 and 2 respectively.P = .297). Women’s range age was 25–46 and 26–44 in groups 1 and 2.Women’s age in group 1 (36.18 ± 3.92) and group 2 (35.88 ± 3.88) was not significantly different (P = .055). A total of 624 warming cycles (n = 2564) were performed, 354 (n = 1469) and 270 (n = 1095) in groups 1 and 2 respectively. There was no difference in the mean number of warmed oocytes with the closed or the open system (4.15 ± 1.65 vs. 4.06 ± 1.41) (P = .147). Survival rate was 70.3% versus 73.3% (P = .096) in groups 1 and 2 respectively. A total of 1386 oocytes (2.23 ± 0.89) were microinjected, 792 (2.25 ± 0.95) and 594 (2.20 ± 0.82) in groups 1 and 2 respectively (P = .304). Fertilization rate (70.8% vs. 74.9%) and the cleavage rate (90.6% vs. 90.3%) showed no difference between the compared groups.A total of 4029 MII oocytes were vitrified. The closed vitrification system was used for 1980 oocytes, while 2049 were vitrified with the open system. There was no difference in the mean number of oocytes vitrified with the closed or the open system (5.09 ± 3.09 vs. 5.30 ± 3.80) (P = .402). Moreover, there was no statistically significant difference between the closed and open groups in the pregnancy rate/transfer (32.0% vs. 31.8%), in the implantation rate (19.7% vs. 19.9%), or in the miscarriage rate (22.1% vs. 21.5%).A total of 297 and 248 transfers were performed in group 1 and 2 respectively. The mean number of embryos transferred was similar in both groups (1.44 ± 0.92 vs. 1.49 ± 0.86)  have impelled scientists to look for solutions that would maintain vitrification in aseptic [The development of a reliable and safe aseptic (closed) vitrification protocol is very important for human cell cryopreservation, especially after the Bielanski reports on the possibility of cross-contamination under LNrotocols –45. So fntainers , 51. In  aseptic , 53. The aseptic , 55.The policy of the University of Bologna Infertility and IVF Unit is to inseminate a limited number of oocytes and avoid the development of supernumerary embryos. Therefore, oocytes’ cryopreservation is offered to most patients.The present study was able to show that there is no significant difference in survival rate between the devices: 70.3% in the closed HSV device versus 73.3% in the open Cryotop device. When compared to two other prospective randomized studies comparing open and closed devices , 57, thiThese results are in line with the study by Papatheodorou: 36.0% clinical pregnancy rate per cycle in the closed Vitrisafe device and 28.0% in the open Vitrisafe device, while Paffoni’s study reported a very low clinical pregnancy rate per cycle of 7.8% in the closed CryoTip device and 26.4% in the open CryoTop device. In the mentioned reports , 58, theAlso, the studies by Papatheodorou and De Munck were conducted on sibling oocyte in a donation program.On March 11, 2020, the World Health Organization (WHO) declared that the COVID-19 infection became pandemic due to its global geographical distribution . Since t.The results of the present study indicate that the high-security vitrification system is efficient and represents a valid strategy for storing biological samples in the safest way to reduce the potential risk of viral contamination. These results are of pivotal importance since many uncertainties remain around the effects of SARS-CoV-2 infection on ART


Treculia africana), African yam bean (Sphenostylis stenocarpa) seed, bambaranut (Vigna subterranean L.), red bean (Phaseolus vulgaris), groundnut (Arachis hypogea L.), African oil bean (Pentaclethra mycrophylla Benth.) seed, cowpea (Vigna unguiculata (L.) Walp) and pigeon pea (Cajanus cajan). The proximate composition, mineral content, fibre profile, fatty acid profile and amino acid compositions were evaluated using standard methods. The results showed that legume samples vary significantly (p < 0.05) in the chemical parameters evaluated. Groundnut, African oil bean seed and African breadfruit had significantly higher protein, carbohydrate, fat and ash contents than other legumes. Equally, groundnut, African oil bean and African breadfruit showed superiority in mineral and fibre abundance, while bambaranut had the lowest mineral and fibre contents. Linolenic acid is the most abundant fatty acid in all the legumes with values ranging from 38.78 – 84.57%. The percentage polyunsaturated fatty acid (PUFA) for all the samples ranged from 40.15 – 48.97%. The total essential amino acids ranged from 24.11 – 66.67 mg/100 g. The range is considered adequate for ideal protein food. Therefore, lesser legumes evaluated can serve as alternative protein sources with good minerals, fibre, essential fatty and amino acids contents.This study evaluated the nutritional composition of Nigeria's lesser known legumes namely African breadfruit ( Food science; Food analysis; Nutrition; Natural product chemistry; Lesser legumes; Minerals; Fibre; Fatty acids; Amino acids. In the tropics, legumes are the second most important food crops after cereals and are excellent sources of cheap plant proteins and minerals when compared with animal products . IndigenLegumes have a special place in the diet of humans, because they contain nearly 2–3 times more proteins than cereals depending on the type . LegumesTo be able to feed the rapidly increasing population in Nigeria, there is need to nutritionally characterize lesser known legumes. Mallotus subulatus (red variety). While, In that regard, research studies have been ongoing in presenting lesser known legumes and their suitability in different food applications. 22.1Treculia africana) seeds, African yam bean (Sphenostylis stenocarpa) seed, bambaranut (Vigna subterranean L.), red bean (Phaseolus vulgaris), groundnut (Arachis hypogea L.), African oil bean (Pentaclethra mycrophylla Benth.) seed, cowpea (Vigna unguiculata L.) and pigeon pea (Cajanus cajan) were procured in the month of January, 2018 from Umuahia Local Market, Abia State, Southeastern Nigeria. The seeds were botanically identified by the Department of Crop Production, Federal University of Technology, Minna, Nigeria. Extraneous matters such as insect infected seed, sand and chaff were manually removed from the samples. Whole seeds of African yam bean, bambaranut, red bean, groundnut, cowpea and pigeon pea were milled in a disc attrition mill  and sieved to a particle size of 1 mm. For African oil bean, the seed coats were removed manually prior to milling. While for African breadfruit seeds, the method of African breadfruit (33.13.1.1The percentage moisture content was determined according to the method described by 3.1.2Fat content was determined using Soxhlet solvent extraction method. Two gram of the sample labelled A was weighed into the extraction thimble and the thimble was blocked with cotton wool. It was then placed back in the Soxhlet apparatus fitted with a weighed flat bottom flask (B) which was filled to about three quarter of its volume with petroleum ether with boiling point of 40 to 60 °C. The extraction was carried out for a period of 4–8 h for complete extraction. Petroleum ether was removed by evaporation on water bath and the remaining portion in the flask was removed along with water during drying in an oven at 80 °C for 30 min. Defatted sample was then cooled in a desiccator and weighed (C). The percentage fat was calculated using 3.1.32SO4/CuS04 and 30 ml concentrated sulphuric acid were added and swirled gently on a heater. The mixture was heated gently at first until frothing stops, then more strongly until a near clear solution resulted. The digested solution was allowed to cool and transferred into a 250 ml volumetric flask. The distillation apparatus was then steamed for 10 min. While this was going on, the volume of the digested solution was made up to the mark and vigorously shaken. Twenty five (25) ml of the digested sample was transferred into Kjeldahl flask and mixed with 25 ml of 40% sodium hydroxide. The mixture of the solution was mounted unto the distillation unit and heated with constant flow of water and the liberated ammonia was collected with 10 ml boric acid-indicator mixture in a conical flask placed at the condenser section of the Markham distillation unit. When the boric acid-indicate mixture turns green, the distillation was allowed to go on for another 5 min. At the end of the time, the conical flask was removed and titrated with 0.01 N hydrochloric acid until the original colour of the boric acid-indicator mixture was restored and percentage nitrogen was quantified using Two gram of the dried sample was transferred into a Kjeldahl flask and 4 g mixture of Na3.1.4The weight of the crucible dish was determined. Two gram of the sample was added to the crucible. The dish and its content were placed on the furnace rake and the furnace temperature was set to 500 °C for 16 h until the sample completely turned into ashes. The crucible dish was removed and kept in desiccator to cool and percentage ash was calculated using 3.1.5Five gram of the sample was weighed into a 500 ml beaker and the content was boiled in 200 ml hydrochloric acid (1%) for 30 min. The suspension was filtered and the residue was washed vigorously with boiling water until it was no longer acidic. The sample residue was then boiled again in a 200 ml sodium hydroxide solution for 30 min, filtered through filter paper (Whatman no.1) and the residue obtained was transferred into a crucible in an air oven 80 °C for 30 min. The dried residue was then cooled in a desiccator and weighed. The weighed sample residue was ashed in a muffle furnace at 550 °C for 30 min. The sample was removed from the furnace when its temperature was 200 °C. It was then cooled in a desiccator and weighed. The loss in weight of the incinerated residue before and after incineration was taken as the crude fibres content and calculated using 3.1.6Carbohydrate was determined by difference using 3.1.7The mineral contents of legume samples were determined as described by The tubes were placed back in the digestion block (second phase of digestion) and left until ca. 1–1.5 ml remains, and then removed from the digestion block. The tubes were checked every 10–15 min during this digestion to avoid drying off. Upon attainment of the required stated mile (1–1.5), the tubes were removed cooled and carefully ca. 2 ml concentrated nitric acid was added and continued heating sustained. The digestion block was turned off when all the tubes have been digested and then removed. The samples were filtered using Whatman hardened ashless #540 filter paper into container appropriate for analysis by AAS. For example for sodium (Na) quantification using AAS, ashed sample was diluted with distilled water to 25 ml, then 0.2 ml diluted to 100 ml. The minerals  were estimated using an atomic absorption spectrophotometer . Phosphorus (P) was measured by converting phosphates into phosphorus molybdenum blue pigment and measured at 700 nm.3.1.8The method of 3.1.93 in methanol at 37 °C for 20 min  and hydrolyzed with 1 ml of 1 M KOH in 70% ethanol at 90 °C for 1 h. The reaction mixture was acidified with 0.2 ml of 6 M HCl and then 1 ml of water was added. FFAs released were extracted with 1 ml of hexane. After evaporation of the hexane in vacuum, the FFAs were methylated with 1 ml of 10% BFr 20 min . The fat3.1.10The method described by 3.2The data obtained were in triplicates and the results were subjected to one-way analysis of variance and expressed as mean with standard deviation. The differences between means were separated by Duncan's Multiple Range Test using IBM SPSS Statistics Programme, Version 19.0 . Significant differences were expressed at 5% level.44.1The protein content of African breadfruit obtained in this study 17.97% is high compared with 10.06% and 12. 27% as reported by 4.2The mineral composition of legume samples is shown in Potassium (K) was the most abundant macro element in all the legume samples evaluated. The values ranged from 844.53 to 1694 mg/100 g in groundnut and bambaranut, respectively. Legume samples evaluated have appreciable abundance of other macro elements Ca, Mg, Na and P. Their values ranged from 185.32 – 415.64 mg/100 g, 122.55–195.92 mg/100 g, 177.65–336.53 mg/100 g and 131.65–526.75 mg/100 g, respectively. Mn is the most abundant trace element determined in the legume samples. The values ranged from 35.26 to 95.39 mg/100 g in groundnut and bambaranut, respectively. Other trace elements Fe, Cu and Zn were present in appreciable quantities ranging from 4.06 – 17.93 mg/100 g, 2.46–11 85 mg/100 g and 15.25 and 41.04 mg/100 g, respectively. However, Sn is the least abundant trace element evaluated. The values ranged from 0.31 to 0.82 mg/100 g in bambaranut and groundnut, respectively.Calcium (Ca) is the most abundant macro element in the human body. In conjunction with phosphorus, they play a role in the process of teeth and bone formation, muscle physiology as well as in the mechanism of blood coagulation . The Ca Magnesium (Mg) is an essential macro element needed for normal muscle and nerve functions; regulation of normal blood pressure and blood glucose level and an important element in teeth and bone formation . LegumesPotassium (K) is an important element which helps in maintaining body fluid electrolytes balance. In association with sodium ions, potassium plays an important role in the brain and nerve functioning and in muscle development. The range determined 844.50–1694.36 mg/100 g shows that all the legume samples are good food sources. The value in groundnut (1694.36 mg/100 g) is high compared with 658.00 mg/100 g in Indian red groundnut variety . The potLegume samples have considerable amount of iron ranging from 4.06 – 17.93 mg100 g. This shows that all the legumes evaluated are potential sources of iron when consumed in sufficient quantity. Therefore, they can serve as important tools in fighting iron deficiency most especially in the developing countries. The iron content in groundnut (17.93 mg/100 g), African oil bean seed (15.67 mg/100 g) and African breadfruit (12.35 mg/100 g) strongly agree with Copper and zinc are important trace elements which play vital roles in the body during metabolisms. They serve as cofactors to a number of key metabolic enzymes . Also, tSelenium (Se) is an essential trace element and it serves as a strong antioxidant which helps in the prevention of mutation and possible cancer development . Further4.3The interaction of dietary fibre with other bioactive constituents such as polyphenol significantly influence their physiological benefits, besides their fermentation in the large intestine which generates short chain fatty acids that serve as microbial fuel .The fibre profile of legume samples is shown in 4.4The result of the essential fatty acids composition of the legumes is shown in Linoleic acid is the most abundant fatty acids in the legumes evaluated and the value ranged from 38.78 - 84.57 g/100 g in groundnut and bambaranut, respectively. This agrees with The total fatty acids (TFA) of the legume samples evaluated ranged from 130.62 – 548.11 mg/100 g. The percentage concentrations of total saturated fatty acids (%TSFA) and total unsaturated fatty acids (%TUFA) ranged from 35.87 – 45.87% and 54.13–65.61%, respectively. The result clearly shows that all the legumes evaluated have high percentage total unsaturated fatty acids with groundnut (54.13%) having the lowest value while, AOB (64.13%) having the highest value. African oil bean had the highest percentage total monounsaturated fatty acids (%MUFA) (18.53%). This was followed by bambaranut, red bean, groundnut and pigeon pea which had 14.61%, 14.56%, 13.98% and 13.80%, respectively. However, African breadfruit had the lowest percentage mono unsaturated fatty acids (12.32%). Evaluating the percentage polyunsaturated fatty acids (%PUFA) of the legume samples, it can be deduced that the values are in the same range. However, bambaranut (48.97%) had the highest percentage while groundnut (40.15%) had the lowest percentage. The range obtained in this study is higher than 37.2% in pumpkin ; however4.5The result of the amino acid profile  shows thGlutamic acid is the most abundant amino acid in all the samples evaluated with groundnut having significantly (p < 0.05) high value (13.29 mg/100 g), while red bean had the lowest value (4.14 mg/100 g). The abundance of glutamic amino acids in legume samples evaluated agrees with the findings of Lagenaria siceraria seed flour and nine underutilised legumes of South West Nigeria. Legume samples evaluated showed good spread of all the essential amino acids. However, sulphur containing amino acids methionine and cysteine were low in abundance with values ranging from 0.86 – 2.78 mg/100 g and 0.56–4.65 mg/100 g, respectively. Sulfur containing amino acids are the limiting amino acids in legumes. Their ranges obtained in this study are high compared with 0.11 mg/100 g in chick pea field pea, green pea, lentils and common beans  contents were between 47.40 – 122.88 mg/100g. The total essential amino acids (TEAA) of the legume samples ranged from 23.29 – 56.21 mg/100 g. The range of total essential amino acids obtained in this study is low compared with 89.00–236.00 mg/100 g in seven important legumes, however, compares favourably with 45.53–48.44 mg/100 g in nine underexploited legume indigenous to South West Nigeria . The ran5Lesser known legumes evaluated differ significantly in their chemical and nutritional compositions. Groundnut, African oil bean seed and African breadfruit showed superiority in protein, carbohydrate, fat and ash contents. Also, in minerals and fibre abundance, groundnut, African oil bean and African breadfruit had high contents while, bambaranut showed the values. In fatty acid composition, linolenic acid is the most abundant fatty acid in all the legumes with values ranging from 38.78 – 84.57%; while, the percentage polyunsaturated fatty acid (PUFA) for all the samples ranged from 40.15 – 48.97%. The total essential amino acids ranged from 24.11 – 66.67 mg/100 g. The range is considered adequate for ideal protein food. Glutamic acid is the most abundant amino acid in all the samples evaluated while, leucine is the most abundant essential amino acid in all the samples evaluated with values ranging from 13.54 to 6.25 mg/100 g.Samaila James: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Titus Ugochukwu Nwabueze, Gregory I. Onwuka: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.Joel Ndife: Performed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.Mohammed Ata’anda Usman: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


The mean correction rate immediately after surgery was 60.8%, and the correction loss rate at the observation end point was 4.8%. The Cobb angle of the lumbar curve was significantly improved immediately after surgery, and the correction persisted until 10 years post-operatively. Remarkable gains were observed for most Scoliosis Research Society-22 patient questionnaire sub-scores at the final follow-up versus preoperative assessments. In conclusion, good correction of the AIS deformity by skip pedicle screw fixation was well maintained over a long follow-up period of 10 years, with clinically meaningful gains in Society-22 patient questionnaire sub-scores.Skip pedicle screw fixation for adolescent idiopathic scoliosis (AIS) requires fewer screws and can reduce the risk of neurovascular injury as compared with segmental pedicle screw fixation. However, the long-term impact of screw number reduction on correction and clinical results is unclear. This study examined the 10-year post-operative outcomes of skip pedicle screw fixation for patients with AIS. We reviewed the outcomes of 30 patients who underwent skip pedicle screw fixation for AIS. Radiological and clinical findings were assessed before and immediately, 2 years, and 10 years after surgery in the remaining 25 patients. The mean Cobb angle of the main curve preoperatively and immediately, 2 years, and 10 years post-operatively was 59.4°, 23.4°, 25.8°, and 25.60°, respectively, and was significantly improved at all post-surgical time points (all  Segmental pedicle screw fixation using numerous thoracic pedicle screws as described by Suk et al.  is a useThe purpose of this study is to examine if the corrective position retention and good clinical outcomes of skip pedicle screw fixation are maintained even at 10 years after surgery. To further validate the usefulness of skip pedicle screw fixation for AIS, we examined the radiological and clinical findings of patients after a long observation period of at least 10 years.doi.org/10.5281/zenodo.4295859).This study was conducted at our institution. We earlier described the 2-year results of skip pedicle screw fixation in 62 consecutive cases of AIS . Among 3Pedicle screw insertion into the upper and lower ends of the instrumentation area was performed bilaterally. Screw placement in the vertebral levels other than the upper instrumented vertebra, lowest instrumented vertebra, and apex was judged based on the size and rigidity of the curve and was skipped when possible. Screw insertion was skipped near the apical vertebrae of each curve and in the junctional zone between structural and compensatory curves that were not close to fused end vertebrae. We excluded pedicles with an outer diameter of less than the thinnest screw diameter during navigation planning. Pedicle screws were routinely inserted near the concave apical vertebrae. For cases in which a 4.5 mm-diameter pedicle screw appeared difficult to insert in navigation planning, tape was used instead. However, in cases where only one vertebra was skipped, no tape was used. Since pedicle diameter is wider near the convex vertebrae, we basically used pedicle screws and skipped one vertebra per inserted vertebra. Screw holes were made using a Kotani probe  with CT-p-value of <0.05 was considered statistically significant.The first author (M.U.), who was not associated with any surgery in the cohort, performed the evaluation. Cobb angle  and thorThe cohort’s preoperative radiological and clinical features are summarized in p < 0.001)  , Table 2p < 0.05)  , Table 2The Cobb angle of the lumbar curve and LIV tilt were significantly improved immediately after surgery, which persisted until 10 years post-operatively , Table 2p < 0.05)  . At the  < 0.05) .p = 0.004). The JOABPEQ scores for low back pain in patients with and without lumbar decompensation were comparable, with values of 91.4 ± 19.2 and 85.7 ± 21.8, respectively (p = 0.58). The JOABPEQ score for low back pain in patients with adding-on was significantly higher than in patients without it . The respective JOABPEQ scores for low back pain in patients with and without early degeneration were similar, corresponding to 62.0 ± 32.9 and 90.2 ± 17.4 (p = 0.27). The mean VAS score for low back pain was 19.6 ± 5.4 (range: 0−80) and was zero in 13 (52%) patients. Bone union was confirmed by CT in all cases. Screw breakage without symptoms requiring surgical revision was seen in two patients. No severe neurovascular complications from surgical invasion or indication for surgical revision were recorded.JOABPEQ scores at 10 years after surgery were 86.8 ± 4.2 for pain-related disorder, 94.0 ± 2.4 for lumbar spine dysfunction, 95.8 ± 2.7 for gait disturbance, 84.4 ± 3.6 for social life disturbance, and 72.9 ± 3.4 for psychological disorder . Three .Ishikawa et al., reported that the final Cobb angle of the thoracolumbar/lumbar curve was significantly correlated with the immediately post-operative LIV tilt . On the The JOABPEQ domain scores in our cohort were all considerably higher than the reference values of low-back-pain patients  Table 4Table 4. Regarding long-term radiological results, the main thoracic curve Cobb angle improved from 51° to 16°, with a correction loss rate of 5% in a five-year study on segmental pedicle screw fixation for thoracic scoliosis . HoweverWith regard to the long-term clinical results of surgical AIS treatment, there are several reports on the Harrington method, posterior fixation with the rod and hook system, and anterior fusion, and few on pedicle screw fixation. In a comparative control study using the SRS-24, AIS patients undergoing correction surgery by the Harrington method had findings equivalent to those of healthy subjects for all parameters, and the magnitude of the scoliotic angle was not associated with quality of life (QOL) scores . In a siWe lastly examined whether the SRS-22r score changes in our cohort were clinically meaningful. In earlier studies on MCID for clinical domain outcomes, MCID was 0.20 for pain (area under the receiver operating characteristic curve (AUC) = 0.723), 0.08 for function (AUC = 0.648), and 0.98 for self-image (AUC = 0.629) . The chaNewton et al., noted that posterior segmental instrumentation for AIS could significantly decrease thoracic kyphosis . Such a The main limitations of this study were no control group, a limited sample size, and a retrospective design. No blinding of the assessor was also a limitation of this study. Although five patients were lost to follow-up, the resulting 83.3% follow-up rate was considered satisfactory based on Solberg et al., reporting that a follow-up loss of 22% did not bias conclusions about the effects of treatment . Thus, oSkip pedicle screw fixation for AIS provided significant and sustained radiological and clinical improvements at 10 years after surgery without serious complications.