Food insecurity affects millions of Americans and college students are especially vulnerable. Little is known about the relation of food insecurity with weight status and dietary intake during this critical phase of emerging adulthood.We aimed to examine the sex-specific associations of food insecurity with obesity and dietary intake among college students. The study also explored these associations by meal plan (MP) enrollment.This cross-sectional study included 683 second-year students at 8 universities in the United States. Food security status and dietary intake were assessed using the USDA Adult Food Security Survey and the Dietary Screener Questionnaire, respectively. On-site anthropometrics were measured by researchers.The prevalence of food insecurity at the universities ranged from 19.0% to 34.1% with a mean of 25.4% for the entire sample. Compared with high food security, marginal food security and food insecurity were associated with 3.16  and 5.13  times increased odds of obesity, respectively, exhibiting a dose–response relation. Food insecurity remained a significant predictor of obesity among both sexes after adjusting for sociodemographic variables. Food-insecure (FI) students had a significantly lower intake of fruits and vegetables and higher intake of added sugars than food-secure (FS) students. Obesity rate and added sugars consumption were higher among FI students with MPs than among FI students lacking MPs and FS students regardless of MP status. Among students with MPs, FS students had a higher intake of fruits and vegetables than FI students.Food insecurity was associated with obesity and poor dietary intake among both sexes. Although MP subsidies may be a reasoned approach to combat food insecurity, it should be coupled with efforts to assist students in making healthy food choices.clinicaltrials.gov as NCT02941497.Registered at  Food insecurity is associated with obesity and poor diet habits among male and female college students. Obesity rate and added sugars intake were highest among food-insecure students with meal plans. Reducing obesity in college students may be possible if specific economic stressors are identified and addressed by applicable social policies. Prior research has identified food insecurity, defined as limited access to adequate and safe foods , as one The association of food insecurity with obesity was first proposed in 1995 by Dietz  who suggDespite a growing body of evidence to indicate a link between food insecurity and obesity, studies among college students have yielded mixed results , 13. Pri1) examined the association between food security status and odds of obesity [defined as BMI (in kg/m2) ≥30] stratified by sex; 2) compared the differences in anthropometric and dietary variables by food security status, and, lastly, 3) explored the difference in dietary variables and weight status by MP enrollment among FS and FI students.To investigate these dynamics, we This cross-sectional study was a secondary analysis of data collected at 8 universities  across the United States under a USDA-funded research project called Get Fruved (NCT02941497). Data collection took place during April 2017 on a sample of second-year students who were invited for an in-person assessment. Participants completed a battery of health-related questionnaires delivered through a web-based format and had their anthropometrics measured by trained researchers. The University of Tennessee Institutional Review Board reviewed and provided ethical approval for all study activities at West Virginia University, South Dakota State University, University of Maine, Syracuse University, and the University of Tennessee. The Institutional Review Boards at the University of Florida, Auburn University, and Kansas State University reviewed and approved the study for their respective campuses, and written informed consent was obtained from all participants before completing the questionnaire and assessment procedures.In accordance with the design of the parent study, participants were eligible based on eating <2 cup equivalents (CE) of fruit and/or <3 CE of vegetables per day as measured by the National Cancer Institute's (NCI's) 9-item all-day screener  and haviFood security was measured using the 10-item validated Adult Food Security Survey (AFSS) developed by the USDA . The que2, and obesity was defined as BMI ≥30. Waist circumference (WC) was measured to the nearest 0.1 cm using a Gulick tape measure  at the midpoint between the bottom of the rib cage and the top of the iliac crest, and hip circumference (HC) at the largest circumference of the hips. Both WC and HC were recorded to the nearest 0.1 cm. Neck circumference (NC) was also measured as it has been associated with obesity-related conditions and chronic diseases , age (18–19/20–21 y), marital status, housing (on-campus/off-campus), race/ethnicity , and parental education (high school or lower/some college or higher). Economic variables included Pell Grant receipt (receives Pell Grant/does not receive Pell Grant), employment (employed/unemployed), and MP (has an MP/does not have an MP).Descriptive statistics were used to report the prevalence of food insecurity, weight status, and the distribution of other participant characteristics, either as frequencies and proportions for categorical variables or as means with SEs for continuous variables. Using logistic regression models, crude and adjusted ORs were reported for associations between food insecurity and sociodemographic characteristics. The outcome in these models was food insecurity, which was recoded into 2 categories: 0 = FS compared with 1 = FI.t tests or Mann–Whitney U tests  were used. Adjusted and unadjusted multiple logistic regression models were conducted to examine the association between food security status and obesity. This analysis was done using a 2-level coding and 3-level coding of food security status. In the adjusted analysis, models were performed including all sociodemographic variables that were significant in the bivariate analysis or hypothesized to influence food security status and obesity. Results from the logistic regression models were expressed as ORs with 95% CIs.To analyze the bivariate association of anthropometric and dietary variables with food insecurity, both in aggregate and by gender, P < 0.05), the Tukey test was used for post hoc comparisons to identify the groups that differed.Finally, to examine the statistical differences in anthropometric measurements and dietary intake of FS and FI students with and without MPs, 1-factor ANOVA  was performed among the following 4 groups: FS with MP, FS without MP, FI with MP, and FI without MP. When ANOVA indicated significant differences , female (69.6%), and non-Hispanic white (47.8%). Around half (52.4%) of the students resided off-campus, almost three-fifths (59.6%) were enrolled in a MP, and almost two-thirds were employed either part time or full time (63.0%). The distribution of student enrollment was as follows: University of Florida, 32.2%; Syracuse University, 14.7%; University of Maine, 14.1%; University of Tennessee, 13.1%; Kansas State University, 10.7%; West Virginia University, 6.6%; South Dakota State University, 4.2%; and Auburn University, 4.2%.Using simple logistic regression analysis, the correlates of food insecurity were housing, employment, Pell Grant, and MP status  of the respondents were FI (P = 0.001). Female FI students had a significantly higher WC , HC (104.0 ± 1.0 compared with 99.0 ± 0.4 cm), and BMI (26.0 ± 0.5 compared with 23.9 ± 0.2) than their FS counterparts. There were no anthropometric differences in male students.Significant associations were noted when comparing FS with FI students on anthropometric variables (P = 0.001) than FS students. On the other hand, FI students had higher intakes of sugar from SSBs  and total added sugars than FS students . These dietary differences were consistent across both sexes.Differences in food consumption of FS compared with FI students were also observed . Among tThe trend in obesity increased with the degree of food insecurity (1) FS with an MP, 2) FS without an MP, 3) FI with an MP, and 4) FI without an MP  = 7.27, P < 0.001], WC , and HC . A Tukey post hoc test revealed that FI students with MPs had a significantly higher BMI, WC, and HC than FS students with and without MPs. By the same token, a significantly higher proportion of FI students with MPs had obesity than of those without MPs (25.3% compared with 12.2%) , whereas students who were both FS and lacking an MP had the lowest prevalence of obesity (4.6%). With respect to dietary intake, significant differences were found in the consumption of fruits and vegetables , sugars from SSBs , and total added sugars . Specifically, FI students with MPs had a significantly lower intake of fruits and vegetables than FS students with MPs. However, there was no difference between FI students with MPs and those without MPs. FI students with MPs had the highest intake of added sugars and FS students without MPs had the lowest intake. No between-group differences were found in the consumption of dairy products, whole grains, calcium, or fiber.To determine the differences in weight status and dietary variables by MP enrollment and food security status, 1-factor ANOVA was conducted across the following 4 groups: The purposes of this study were to examine the sex-specific associations between food insecurity and obesity among college students and to compare the differences in dietary intake between FS and FI students. An exploratory aim of this study was to assess the differences in weight status and dietary intake by MP enrollment. Results suggested that college students are at risk of food insecurity and justify calls for interventions aimed to mitigate its burden. Twenty-five percent of students from this sample reported food insecurity. In addition, 22.3% were classified as marginally FS, displaying anxiety over food sufficiency. Consistently across both sexes, students with food insecurity were at higher risk of having obesity and poorer dietary intake than their counterparts. Although additional studies are needed to understand the observed associations, an exploration of weight status and dietary intake by MP enrollment showed a higher intake of added sugars and higher rates of obesity among FI students with MPs than among FI students without MPs and FS students regardless of MP status.Food insecurity was positively related to obesity in a dose-response pattern even after adjusting for potential confounders. Compared with students who were highly FS, students with marginal food security and those with food insecurity were 3 and 5 times more likely to have obesity, respectively. Although “marginal food security” has been classified as “food security” in the US government's estimates, college students who are marginally FS may be more similar to FI students than to FS students in their sociodemographic characteristics, psychosocial profiles, and patterns of disease . Indeed,Contrary to previous studies of nonstudent adults reporting an association between food insecurity and obesity only among women , 29, fooThe mechanisms for the association between obesity and food insecurity are still not well elucidated. One proposed mechanism is that FI individuals may consume nutritionally inadequate diets, yet still have an energy intake that exceeds their daily requirements . LackingThis study identified the need for further exploration of MP subsidies as a mechanism to conjointly address food access and promote healthy food choices. The rate of obesity and added sugars intake were higher among FI students with MPs than among FI students lacking MPs. It is noteworthy, however, that MPs are defined differently on each campus varying from partial to all-you-can-eat, and these differences were not captured in the current study. FI students may alternate between cycles of scarcity and availability depending on the type of MP in which they are enrolled. From the current exploratory findings, it is plausible that FI students with access to the dining halls engage in an adaptive “buffering” behavior against any future uncertainty when MP benefits are running low. This observation is comparable with the pattern observed linking food assistance programs and obesity, in which the relation between these programs and obesity is greatest in those who are FI . The “foFindings from the present study support theories suggesting the presence of underlying psychological and physiological processes that drive decisions toward energy-dense food choices and subsequent fat deposition in FI individuals . When coInterpretation of these findings necessitates consideration of study limitations. Although the study's cross-sectional nature allowed for the ascertainment of associations and development of hypotheses concerning relations between food insecurity and obesity, it is not possible to establish causality using this design. The use of longitudinal designs with repeated measures of obesity and food insecurity can provide an indication of causal pathways but experimental studies are necessary to establish causality.Our findings may not apply to all US college students. Because the parent study was designed to include students at risk of poor health behaviors, the nature of this sample might have influenced our findings of the relation between food insecurity and obesity. Likewise, although our sample was drawn from 8 states, it was limited to those in the Eastern part of the United States, especially the Southeast. Other regions like the Midwest and West were not represented, and these tend to have lower proportions of food insecurity and obesity than others , 40. ThuAlthough widely used in this population, the USDA AFSS has not been validated in college students. This factor may have introduced survey response error because it is unclear whether students interpret and respond to the questions like other populations. In addition, there have been suggestions that food insecurity experienced during childhood and adolescence has effects on weight gain later in life , and theIn conclusion, this study was the first to examine the sex-specific association of food insecurity with obesity among college students and explore the differences in obesity and dietary intake by MP enrollment. A relatively high prevalence of food insecurity was reported among this sample of college students. Furthermore, both male and female FI students had an elevated risk of obesity and tended to have less desirable dietary outcomes than their FS counterparts. Access to MPs may help improve food access, because students without MPs had greater odds of reporting food insecurity. On the other hand, FI students with MPs had the highest rates of obesity and added sugars intake. Subsidizing dining for students experiencing immediate food access issues and those at risk of food insecurity should be coupled with educational programming to improve the consumption of fruits and vegetables and reduce the intake of added sugars, specifically from SSBs. Continued efforts to implement and test the effectiveness of such strategies on college campuses are needed to reduce the burden of food insecurity and improve health in college students.


We found that SNR ranged from 4.1 to 9.8 dB, intrasession and intersession ICC values ranged from 0.629 to 0.886, CVs ranged from 0.099 to 0.241, and JS divergence ranged from 0.18 to 0.20, demonstrating high JAE repeatability and signal strength at three locations. The volunteer sample size is not large enough to represent JAE analysis of a larger population, but this work will lay a foundation for future work in using wrist JAEs to aid in diagnosis and treatment tracking of musculoskeletal pathologies and injury in wearable systems.Joint acoustic emission (JAE) sensing has recently proven to be a viable technique for non-invasive quantification indicating knee joint health. In this work, we adapt the acoustic emission sensing method to measure the JAEs of the wrist—another joint commonly affected by injury and degenerative disease. JAEs of seven healthy volunteers were recorded during wrist flexion-extension and rotation with sensitive uniaxial accelerometers placed at eight locations around the wrist. The acoustic data were bandpass filtered (150 Hz–20 kHz). The signal-to-noise ratio (SNR) was used to quantify the strength of the JAE signals in each recording. Then, nine audio features were extracted, and the intraclass correlation coefficient (ICC) (model 3, The wrist is one of the most injured joints in athletes, especially in adolescents. Of all adolescents who participate in athletics, 1.3% have sustained wrist injuries via traumatic injuries in contact sports and overuse injuries in golf, racquet sports, and gymnastics . FurtherRecent work has demonstrated the use of inexpensive accelerometers to noninvasively aid in joint health assessment by sensing the vibration on the surface of the skin—termed “vibroarthrographic” signals , or moreThis study followed the Guidelines for Reporting Reliability and Agreement Studies (GRRAS) . All humSeven healthy college-aged volunteers  were recruited. Inclusion criteria for participation in this study dictated volunteers must have no history of major wrist injury or degenerative joint disease. Additionally, if volunteers had changes to wrist joint health between recording sessions, they would be excluded from the study. No volunteers met this exclusion criteria, so no such exclusions were made. Other factors which may affect wrist JAEs such as volunteers’ daily medication usage, day-to-day wrist activity levels, and history of previous minor wrist injuries were not controlled. An additional volunteer who had juvenile idiopathic arthritis (JIA) as a child  was included to facilitate proof-of-concept qualitative comparisons to our healthy volunteers, and to allow the development of hypotheses for future studies to address.A previously recorded dataset  of JAE recordings from knee squat exercises recorded at sites 2 cm medial and lateral to the patellar tendon on both legs of college-aged individuals with no history or major knee joint injury or degenerative disease was also used in order to compare the signal strength measurements attained in the analysis of the recordings from the wrist against the two locations on the knee known to provide salient knee health assessment. The same data acquisition system, preprocessing steps, and signal strength assessment methods were used in this dataset as were used in our wrist recordings to ensure consistency in methods to provide a valid comparison. On each day of participation, volunteers preconditioned their wrists by moving them around for 20–30 s. Volunteers then practiced following the animations which guided them to move their wrist according to the two prescribed exercises at the desired movement speed (2 s cycle period). Then, four contact microphones were attached to four of our eight selected locations shown in  around tOur JAE sensing system consists of four miniature uniaxial accelerometers  with wide bandwidth (2 Hz–10 kHz), and high sensitivity (100 mV/g). The accelerometers, used as contact microphones, were fixed to the skin using double-sided tape . The other end of the accelerometers was connected to a computer via a data acquisition system  recording vibrations at a sampling rate of 50 kHz for subsequent processing using scripts in MATLAB .An inertial measurement unit (IMU)  was connected to a microcontroller  to monitor the motion of the wrist joint relative to the forearm while volunteers performed the wrist exercises necessary to excite JAEs. To best track wrist motion, the forearm was held stationary by strapping it to the arm of the volunteer’s chair and the IMU had to be held in the hand of the wrist in motion. A custom grip shown in  made of The first step in processing the raw audio signal was applying a Kaiser window bandpass filter (150 Hz–20 kHz) to the audio signal recordings. At this point, we performed qualitative analysis by visually comparing the filtered waveforms , the Teager energy operator was used to find the characteristic JAE clicks in the signal, chosen as the peaks greater than 20% of the range of the signal value as has been proven to be successful in JAE analysis by Semiz et al. . Click wk) to calculate ICC values with a 95% confidence interval (CI) [All statistical analysis was performed using scripts in MATLAB. The mean, median, and standard deviation of the features of all windows were calculated and stored as a vector of features for each cycle, where there are 60 such feature vectors for each combination of volunteer, wrist, exercise, and location (six sessions and 10 cycles for each). The average of each feature over those 60 vectors was calculated to give a single averaged feature vector for each combination of volunteer, wrist, exercise, and location. To calculate the intraclass correlation coefficient (ICC) to describe intersession reliability, each feature vector acts as a measurement where every feature is a rater. We used the two-way mixed effects, consistency, multiple raters/measurements model of ICC  . All meaval (CI) , where vTo find the JS divergence, distributions of each feature are needed: the nine features for each bin were kept such that they could be summarized as a histogram of the feature distribution for each recording of 10 cycles. Then, the average distribution of similar recordings on a day  was taken. The KL divergence of each of those similar recordings was calculated using the averaged distribution as the “ground truth recording” ,26. AverThe standard error of measurement (SEm) was found for both JS divergence and SNR calculations. The SEm was the multiplied by 1.96 to give a confidence bound, which was added to the top and bottom of the measurement (JS Divergence or SNR) to give a 95% CI. The coefficient of variation (CV) was also calculated for each feature at each location and exercise to assess intersession and intrasession variability, where low levels of variation are defined as CV values less than 12% .The calculated intrasession ICC values with 95% CIs for flexion-extension and rotation exercises over all tested locations are displayed in Intrasession ICC with 95% CIs for all locations are shown in Assessing the signal strength relative to noise level after filtering, the median SNR was found to be 6.0 dB (95% CI of 5.8–6.2 dB) for flexion-extension and 6.1 dB (95% CI of 5.7–6.5 dB) for rotation with standard deviation between volunteers of 1.9 dB for flexion-extension and 1.0 dB for rotation. p < 0.001, using two-sample t-test with Bonferroni correction). Locations D1, D2, and M1–M3, where median SNRs were 4.5 (95% CI of 3.6–5.3), 5.6 (95% CI of 4.9–6.3), 4.1 (95% CI of 3.6–4.7), 4.2 (95% CI of 3.5–4.9), and 6.3 (95% CI of 5.7–6.9) dB, respectively. Additionally, the standard deviation of SNRs among volunteers ranged from 1.6 to 2.5 dB. The dataset of recordings from knee squats of healthy volunteers showed a median SNR of 5.5 (95% CI of 4.8–6.3) dB at 2 cm medial to the patellar tendon and a median SNR of 7.2 (95% CI of 6.6–7.9) dB 2 cm lateral to the patellar tendon.SNR values for each recording and summarizing by location yields Recordings taken from the wrist joint during the prescribed motions must capture wrist JAEs. The highest quality JAE signals gathered in previous work come from Whittingslow et al.’s study on joint sounds within a cadaver knee, as observed JAEs did not exist in a knee before meniscus tear and meniscectomy. Thus, they could be directly attributed to changes within the articulating surfaces within the knee . For ourp < 0.05 for intersession ICC and p < 0.1 for intrasession ICC, using two-sample t-test with Bonferroni correction), so we may conclude that rotation exercises more reliably create the dynamic interactions within the wrist joint which excite JAEs. Similar repeatability analysis using intrasession JS divergence  on the nine identified features extracted from the filtered acoustic signal confirms the high levels of repeatability also demonstrated by the ICCs. Both exercises showed acceptable levels of signal strength with some variation between the healthy volunteers, but there was not a significantly better exercise for minimizing motion artifacts . However, SNR may be more dependent on microphone location due to motion artifacts, thus eliminating noisier locations would improve the overall performance of each exercise. We determined both unweighted flexion-extension and rotation exercises can repeatably excite and record wrist JAEs with moderate signal strength, and the features of sounds which have performed well in knee joint health classification studies show high repeatability in our recordings. Therefore, we can conclude that both unweighted wrist flexion-extension and rotation exercises are suitable for use in clinical studies in future work. Since both prescribed exercises excite wrist JAEs, we can use JS divergence and intersession and intrasession ICC and CV to assess the repeatability of these exercises exciting wrist JAEs ,19. Our p < 0.001, using two-sample t-test with Bonferroni correction), and locations D2 and M3 showed moderately higher signal strength than the rest of the locations . Additionally, the standard deviation of SNRs among volunteers indicates some variability in noise levels at different locations among volunteers. Though this may alter which location produces the best signal quality for individual volunteers, the overall separation between proximal locations and all other locations indicates that due to the decreased skin motion relative to the underlying skeletal structure [It has been demonstrated that both wrist flexion-extension and rotation exercises consistently excite wrist JAEs which can be picked up by contact microphones placed on the skin. However, some microphone placement locations have higher levels of noise due to motion artifacts. Quantifying this as an SNR value for each recording and summarizing by location yields the results displayed in tructure ,30, thesp < 0.05 for intrasession ICC, using two-sample t-test with Bonferroni correction), and separation decreases so good separation  is only present between the lowest and highest (M3) performing locations for intersession ICC. Additionally, our locations with highest repeatability also showed highest level of noise and motion artifact interference, which we have observed to inflate our consistency measurements, further reducing the true separation between these measures across locations. However, the degree of repeatability shown by the ICC  demofication ,19. p < 0.001, using two-sample t-test with Bonferroni correction), which had relatively weaker SNR scores. Additionally, the strongest signal strength demonstrated at location P1 showed high levels of separation from all other locations . This reveals the best locations for minimizing artifact interference while still having fair repeatability are proximal to the wrist joint, and location P1, which is 3 cm proximal to the wrist joint and centered between the radius and ulna on the dorsal side, is best for minimizing noise and motion artifact levels. On the other hand, locations P1 and P2 have the lowest levels of intersession and intrasession repeatability, which can be attributed to reduced proximity to the wrist joint and the fact that the soft tissue these locations rest upon will not transmit vibrations as well as the harder tissues of other locations, such as locations P3, D1, D2, and M1–M3, which are all clustered with high repeatability levels. The best balance of moderate-to-high signal strength and excellent repeatability of JAE recordings in healthy volunteers can be shown at locations P3, D2, and M3. The combination of fair-to-high repeatability demonstrated by ICC and JS divergence analysis at all locations around the wrist and the acceptable signal strength demonstrated with the SNR at locations P1–P3, D2, and M3 allows us to conclude that locations P1–P3, D2, and M3 are suitable for high quality repeatable wrist JAE recordings. The strong SNR values at locations P1–P3 had high separation over all other locations (n = 7) and the low-to-medium levels of variability between volunteers for our measurements means we cannot know if our sample of healthy volunteers is representative of a larger population with varied wrist joint pathologies. Future studies researching the effectiveness of quantifying wrist joint health using the locations described in this study must first perform qualitative assessments by looking at and listening to the audio signal to identify grinding and clicking sounds characteristic of JAEs to confirm the quality of wrist JAE recordings. Our volunteer sample size  or wrist injuries as has been done in previous studies on the knee. This framework could be used to create techniques for clinical use of wrist JAEs, which may allow for the development of wearables for at-home wrist joint health monitoring. Such technology would allow for quicker diagnosis of wrist joint injuries and chronic joint conditions, and could improve treatment monitoring by providing clinicians with quantitative measurements to help assess patients’ wrist joint health more frequently without the need for additional clinic visits, which would benefit patient outcomes while reducing the burden on the medical systems that treat such injuries and chronic joint conditions. Moreover, given the increased reliance on telemedicine during the current pandemic, technologies for enabling joint health assessment in the home environment are imminently needed to facilitate comprehensive physical examination of joint health in a remote setting.


The findings suggested that cacao bean meal can be used as a feed supplement in diets of broodstock fantail goldfish for improving the growth, health status, and female reproductive performance, economic efficiency, and gonadal histological structure.Recently, the use of medicinal herbs for regulating reproduction has received much attention in aquaculture, as they are safe, effective, biodegradable, and locally available. The data on the use of cacao bean meal as a food supplement for fish are extremely scarce. This study assessed the possible effects of cacao bean meal as a feed supplement on the growth, health status, blood biochemical parameters, antioxidant, immune status, physiological parameters, female reproductive performance, and gonadal histological features of fantail goldfish. The experimental treatments consisted of three levels of cacao bean meal 0, 5, and 10 g kgTheobroma cacao L., (CBM) on the growth, health status, blood biochemical parameters, antioxidant, immune status, physiological parameters, female reproductive performance, and gonadal histological features of fantail goldfish  were evaluated using a complete randomized block design with sex as a block. The trial lasted for 60 days. A total of 54 healthy fantail goldfish  were randomly allocated into three treatments with supplementation of three levels of cocoa powder 0, 5, and 10 g kg−1 diet, CBM0, CBM5, and CBM10, respectively, with the sex ratio being four females:two males per replicate. The body weight gain and feed conversion ratio of males were increased in the CBM10 treatment (p < 0.05). The CBM10 diet improved relative feed costs (p < 0.05). Females fed on the CBM10 diet had an increase in the serum level of total protein (p = 0.001). Females fed on a diet supplemented with CBM5 showed a decrease in the serum level of triglyceride compared to females fed on CBM0 and CBM10 diets (p = 0.03). CBM10 diet increased the serum superoxide dismutase (SOD) activity of fish compared to CBM0 and CBM5 diets (p = 0.004). Serum levels of testosterone and estradiol were significantly increased in males fed on the CBM10 diet. The female reproductive performance was improved by CBM supplementation (p < 0.05). Ovarian histology exhibited increased granulation and follicle numbers after dietary CBM supplementation compared to the control treatment. Therefore, cacao bean meal can be used as a feed supplement in the diets of fantail goldfish for improving the growth, health status, and female reproductive performance, economic efficiency, and gonadal histological structure.The potential effects of cacao bean meal,  Carassius auratus, L.) is one of the most popular fish used as a biological model in many laboratories [−1) can improve the reproductive performance of broodstock goldfish [−1) improved the gonadosomatic index in broodstock goldfish. The study of Kavitha, et al. [(Poecilia latipinna) to Tribulus terrestris extract direct their sex more towards maleness and boosted spermatogenesis. On the other hand, a recent study reported a reduced absolute fecundity, gonadosomatic index, and bad changes in the gonadal histology of Oreochromis niloticus, which includes degenerating seminiferous tubules, few to absence of spermatozoa in the lumen of seminiferous tubules, and loss of the testicular architecture by using Aspilia plant (Aspilia mossambicensis) and Neem tree powders (Azadirachta indica) as feed supplement that was induced by alkali and flavonoids [Carica papaya extract could improve growth and enhance gonadal development in Nile tilapia [Goldfish  as a dietary supplement on the growth, health, and reproductive performance of fantail goldfish. The present study was conducted in one of Abu Sweir city farms, Ismailia Governorate, Egypt. The experiment was performed according to national and international institutional guidelines for the care and use of animals for scientific purposes, and ethical approval was obtained from the sponsoring institute Agriculture Research Center, Egypt (ARCIACUC-2019). The fish did not show any clinical abnormalities and did not have any history of disease outbreaks. The fish health status was checked before the experiment according to the guidelines of Canadian Council on Animal Care, CCAC .A total of 54 healthy fantail goldfish  were obtained from a private fish farm at Abu Sweir city, Ismailia Governorate, Egypt. From May to the middle of July, climatic conditions were suitable for spawning and the average water temperature was 25 ± 1 °C. The fish were kept in nine glass aquaria (60 L) capacity. About 25% of aquarium water (tap water free from chlorine) was exchanged daily. The aquaria were supplied with continuous aeration by air stone and fish were fed with a basal diet for two weeks before the beginning of the experiment. The water quality parameters were kept according to American Public Health Association, APHA  with a cRaw cacao beans were purchased from a local market . The beans were dried in a hot air oven at 105 °C for 3 h, crushed, and ground into a fine powder using an electrical blender, strained through a 0.25 mm sieve, and finally stored in the refrigerator (at 4 °C) in labeled airtight polyethylene bottles until its use. The proximate composition of cacao bean meal was measured according to AOAC , that re−1 diet, CBM0, CBM5, and CBM10, respectively, three replicates for each treatment , with the sex ratio = four females:two males per replicate. The experiment lasted for 60 days. The proximate chemical composition of the basal diet  was extracted using absolute ethanol for 6 h in a Soxhlet, and then the extract was filtered using Whatman filter paper No.1. After cooling the excess of solvents, aqueous and organic extract were removed under vacuum using a rotary evaporator. The extract was stored in the refrigerator (at 4 °C) for further use. The elements C, H, O, N, S were analyzed using the GC- mass techniques at the regional center for Mycology and Biotechnology Al-Azhar University, Egypt. The technique was performed using Trace GC1310-ISQ mass spectrometer  with a direct capillary column TG–5MS (30 m × 0.25 mm × 0.25 µm film thickness). The column oven temperature was initially held at 50 C and then increased by 5 °C/min to 230 °C hold for 2 min, then increased to the final temperature of 290 °C by 30 °C/min and hold for 2 min. The injector and MS transfer line temperatures were kept at 250 and 260 °C respectively; helium was used as a carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 3 min and diluted samples of 1 µl were injected automatically using an Autosampler AS1300 coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 40–1000 in full scan mode. The ion source temperature was set at 200 °C. The components were identified by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 11 mass spectral database.Chromatographic characteristics by GC-mass techniques showing the active principles in cacao bean meal ; dodecanEconomic parameters were calculated according to El-Telbany and Atallah  and DunnTotal costs (USD) = Fixed costs + feed costs. Feed costs (USD) = the cost of one kg of each diet including the cost of cacao bean meal (3.79 USD/kg) × the amount of total feed intake (kg) during the experimental period (60 days).Relative feed cost (USD/kg of weight gain) = total feed costs/total weight gain−1 benzocaine solution  according to Neiffer and Stamper [g. The obtained serum was used for the determination of some blood biochemical indices and the levels of the antioxidant enzymes. The serum was stored at −20 °C in screw cap glass vials until use [At the end of the experiment (60 days), ten fish/treatment  were collected and anesthetized with 100 mg L Stamper , and blontil use .The total protein, albumin was estimated following the technique described by Doumas, et al. . The quaLysozyme activity was measured using fish Lysozyme ELISA kits with CAT. NO. MBS099538 following the instruction of the manufacturer . The activity of myeloperoxidase (MPO) was determined using fish Myeloperoxidase ELISA Kit (My Biosource Co. CAT NO. MBS016324). Serum nitric oxide (NO) was determined using Fish Inducible Nitric Oxide Synthase ELISA Kit (My Biosource Co. CAT NO. MBS023530).Catalase (CAT) activity was measured using fish Catalase ELISA Kit (My Biosource Co. CAT NO. MBS038818). Superoxide dismutase (SOD) activity was evaluated using fish Superoxide Dismutase (SOD) ELISA kit (My Biosource Co. CAT NO. MBS705758). Reduced glutathione (GSH) level was estimated using reduced glutathione (GSH) Assay Kit (My Biosource Co. CAT NO. MBS2540412).The serum level of follicle-stimulating hormone (FSH) was estimated using fish Follicle-Stimulating Hormone ELISA Kit (My Biosource Co. CAT NO. MBS281137). The serum level of Luteinizing Hormone (LH) was estimated using fish Luteinizing Hormone ELISA Kit (My Biosource Co. CAT NO. MBS031319). Testosterone hormone was assessed using fish Testosterone (T) ELISA Kit (My Biosource Co. CAT NO. MBS933475). 17 β estradiol hormone was measured using fish Estradiol ELISA Kit (My Biosource Co. CAT NO. MBS283228).C. auratus was evaluated at two spawning periods; the first spawning was after 10 days from the beginning of the experiment and the second was after 25 days from the first. Carassius auratus eggs look like small round “bubbles”. They are clear in color except for a small dark spot in the middle of the egg. Healthy goldfish eggs look like small, clear bubbles and can range in color from white to yellow-orange. Dead eggs were pale yellow and were removed carefully. The eggs of C. auratus are adhesive and are attached to the roots of floating plants, which is best suited to the large aquarium and used as an egg collector. One of those plants that floated on the surface of the water was Pistias tratiotes, commonly called water lettuce. As soon as spawning was finished, the plants were transferred to a new aquarium with water temperature = 25 °C, where the eggs are hatched (72 h after spawning). Collection of seeds and swim-up fry first appeared during the 10 days of pairing. The seeds  were collected and counted after stocking up. The experiment was completed in 60 days.The reproductive performance of female The average number of eggs per spawning, the average number of fries per spawning, the average weight of eggs, the average weight of fry, embryonic development, and hatching rate of broodstock goldfish were calculated according to Boonyaratpalin .Hatching rate% = no. of egg hatched/total no. of eggs × 100.The average number of eggs per spawning = total number of eggs per tank/number of spawnings.The average number of fries per spawning = total number of fries per tank/number of spawnings.®, Wetzlar, Germany) and stained with hematoxylin and eosin (H&E). Slides were examined and photographed using the AmScope digital camera-attached Ceti England microscope for histopathological examination [At the end of the experiment, 5 females and 3 males from each treatment were sacrificed by pithing, and separating the brain and spinal cord, and gonads were collected. Samples were fixed in 10% neutralized formalin solution, followed by washing with tap water, then dehydrated in ascending grades of ethyl alcohol (70–100%), cleared in xylene, and embedded in paraffin. Tissue sections of 5 µ thicknesses were prepared with the help of microtome (Leicamination . p > 0.05). The data were analyzed using SPSS 18.0 for Windows . Variations were assessed by complete randomized block design one-way (ANOVA) to examine the effect of cacao bean meal on the economic efficiency and female reproductive performance with controlling the effect of the spawning period. A complete randomized block design two-way (ANOVA) was used to examine the effect of cacao bean meal on the growth and blood biochemical parameters of fantail goldfish. Post-hoc Tukey’s multiple range tests were performed to compare the differences between the means at 5% probability. The variation in the data was expressed as the mean ± standard deviation (SD) and the significance level was set at p < 0.05. Shapiro–Wilk’s test was used to verify the normality and Levene’s test was used to verify homogeneity of variance components between experimental treatments and the assumption were achieved (C. auratus is shown in p = 0.001). The FCR was decreased in CBM10 diet compared to CBM0 diet (p = 0.01). Males showed higher FBW and BWG (p = 0.00) and lower FCR (p = 0.00) than females. The interaction between the CBM level and the sex increased the BWG in males fed the CBM10 diet compared to males fed CBM0 and CBM5 diets (p = 0.02). The BWG of males fed on CBM5 and CBM10 diets was increased by 10.21 and 22.06%, respectively. The collective feed intake (137.94 ± 0.66 g/fish) was not significantly affected by CBM supplementation (p > 0.05). The effect of CBM, sex, or their interaction on the growth performance parameters of C. auratus diets is shown in p = 0.02). The relative feed cost was decreased in the CBM10 diet compared to the CBM0 diet (p = 0.02). The relative feed cost was improved by 5.08 and 14.88% for CBM5 and CBM10 diets, respectively. The economic value of using CBM as a feed supplement for C. auratus is shown in p = 0.02). The serum levels of total protein, α2 globulin, and β globulin were significantly higher in males than in females . The interaction between the CBM level and the sex increased the serum level of total protein in females fed on the CBM10 diet compared to females fed on CBM0 and CBM5 diets (p = 0.001). Females fed on the CBM5 diet showed a significant decrease in the serum level of triglyceride compared to females fed on CBM0 and CBM10 diets (p = 0.03). The effect of CBM, sex, or their interaction on the blood biochemical parameters of C. auratus. CBM10 diet increased the serum SOD activity of fish compared to CBM0 and CBM5 diets (p = 0.004). Carassius auratus males had a higher serum level of GSH than females (p = 0.01). No significant effect of the interaction between the CBM level and the sex on the antioxidant and immune status of C. auratus. p = 0.01). Dietary supplementation of CBM decreased the serum level of estradiol of fish compared to the CBM0 diet (p = 0.01). The serum levels of testosterone and estradiol were higher in males than in females . The interaction between the CBM level and the sex increased the serum levels of testosterone and estradiol in males fed on the CBM10 diet compared to males fed on the CBM0 diet .As shown in Carassius auratus eggs hatched from the CBM10 treatment first then eggs from the CBM5 treatment and finally eggs from the CBM0 treatment. The results concerning the effect of CBM on reproductive performance are illustrated in Table 7. The average egg weight, average fry weight, average number of eggs per spawning, average number of fries per spawning, and embryonic development were significantly increased in CBM5–10 treatments compared to CBM0 treatment . The hatching rate% was significantly increased in CBM5 treatment compared to CBM0 treatment (p = 0.01). The ovaries of all the fish fed on CBM diets had the more or less similar architecture of the ovarian follicles, indicating that the supplementation of different levels of CBM was useful for the fish. Ovary of CBM0 treatment showed normal architecture of ovary with normal follicles and granulation A. CBM5-fTestes of fantail goldfish fed with CBM0 showed the normal structure of seminiferous tubules with spermatocytes, spermatids, and few sperms D. CBM5-fC. auratus with diets supplemented with cacao bean meal at a dose of 5 and 10 g kg−1 diet caused no mortalities among all treated groups, which help us to exclude the ID50. The results of the current work explained that diets supplemented with CBM could successfully boost some health parameters in C. auratus. The results indicated that CBM10-supplemented diets improved the growth performance of C. auratus and the interaction between the CBM level and the sex increased the BWG in males fed the CBM10 diet. Uzochukwu [larias gariepinus, fed on a diet supplemented with 10% CBM. The improved growth by CBM supplementation may be a result of the ascorbic acid content of cacao bean meal. L-ascorbic acid is a micronutrient important for normal physiological function and growth of most aquatic animals [Heterobranchus longifilis, by dietary supplementation of ascorbic acid. The current study also showed that the body weight gain in males was higher than females, this may be because a considerable amount of energy in female fish is directed to reproductive activity and egg production as reported by [In aquaculture, the use of medicinal herbs to regulate reproduction has received great attention in recent times as they are safe, effective, biodegradable, and locally available. The data available for using cacao bean meal as a feed supplement for broodstock fantail goldfish is scarce. As an initial experiment, feeding of zochukwu  reported animals , as a re animals . Adeebay animals  showed iorted by .Efforts to find ways to reduce the feed cost for fish production have evaluated a wide variety of feed supplements ,45,46,47Regarding the effect of CBM, sex, or their interaction on the serum biochemical parameters, the interaction between CBM level and sex increased the serum level of total protein in females fed on the CBM10 diet and decreased the serum level of triglyceride in females fed on the CBM5 diet. This indicates the potential of CBM to increase defensive proteins, stimulating the immune response. The high values of blood proteins especially globulins are a good indicator of improved liver functions and innate immune response . This maC. auratus, the CBM10 diet increased the serum SOD activity of fish and the males had a higher serum level of GSH than females, which could be attributed to the difference in the metabolic activity for both males and females [The oxidative stress can destroy many vital biological molecules such as DNA and proteins. There is an antioxidant defense system that protects the fish tissues from oxidative damages . Antioxi females . The ant females . Flavano females . Additio females . Beside, females . Additio females . All vertebrates, including fish species, produce the gonadotropins (GH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) from the pituitary gland and they are the main regulators of gonadal reproduction and development. In fish, early phases of gametogenesis, such as spermatogenesis and vitellogenesis, are regulated by FSH, whereas the final maturation processes, such as ovulation, oocyte maturation, milt production, and sperms production, are the function of LH ,62. The Xiphophorus helleri [Danio rerio) to yield healthy larvae. Energy may be transferred from somatic growth during periods of increased energy demand, such as the end of the gonadal development [Female reproductive performance is strongly influenced by the nutritional status of fish that is well-known to have many reproductive traits, such as fecundity, age at maturity, egg size, embryonic development, and chemical composition of eggs ,73,74. T helleri . Kolb, e helleri  demonstrelopment ,80, sincClarias gariepinus, females fed a diet supplemented with 10% CBM, and increased testicular weight and gonadosomatic index values in males fed a diet supplemented with 10 and 40% CBM. His results obtained for gonadal histology and histomorphology showed more developed gonads in fish fed diets supplemented with 10 and 40% CBM. The improved gonadal architecture in the current study can be attributed to the effect of CBM supplementation on boosting the serum gonadotrophic hormones and 17ß-estradiol hormone. Gonadotropin hormones (LH and FSH) are essential pituitary hormones that regulate the maturation and development of gonads [Anguilla japonica, indicated by increased spermatogonia number by dietary ascorbic acid addition.Regarding the histological features of the gonads, the results exhibited increased granulation and numbers of follicles, and better effect towards fecundity and granulation after the addition of various concentrations of CBM. In the case of the testis, the histological features of all treatments were within the normal range. The study of Uzochukwu  indicatef gonads ,82,83. Ff gonads . The 17ßf gonads ,86. The f gonads . Additiof gonads  who obseThe current study demonstrated that cacao bean meal can be used as a feed supplement in diets of fantail goldfish for improving the growth, health status, female reproductive performance, and gonadal histology of fantail goldfish. Additionally, supplementing the diets of fantail goldfish with cacao bean meal improves the relative feed cost; however, further studies are recommended to assess the inclusion of higher levels of CBM and to investigate the immunomodulation and other actions induced by CBM in different fish species.


The implantation rate and the live birth rate per cycle were comparable between the two groups (p = 0.05), but the cumulative live births rate was significantly lower in in the endometriosis group , as a consequence of the lower number of frozen embryos. In conclusion, endometriosis lowers the cumulative live birth rates by decreasing the number of embryos available to transfer, but not their quality.Endometriosis and infertility are closely linked, but the underlying mechanisms are still poorly understood. This study aimed to evaluate the impact of endometriosis on in vitro fertilization (IVF) parameters, especially on embryo quality and IVF outcomes. A total of 1124 cycles with intracytoplasmic sperm injection were retrospectively evaluated, including 155 cycles with endometriosis and 969 cycles without endometriosis. Women with endometriosis had significantly lower ovarian reserve markers (AMH and AFC), regardless of previous ovarian surgery. Despite receiving significantly higher doses of exogenous gonadotropins, they had significantly fewer oocytes, mature oocytes, embryos, and top-quality embryos than women in the control group. Multivariate analysis did not reveal any association between endometriosis and the proportion of top-quality embryo (OR = 0.87; 95% CI [0.66–1.12];  Endometriosis is a complex gynecological disease whose pathogenesis is still widely debated. It can affect several anatomical sites such as the ovaries, the peritoneum, the bladder and the rectum. This disease is associated with infertility, dysmenorrhea, and chronic pelvic pain and can impair quality of life in more than one way ,2,3. TheWe performed a retrospective cohort study based on all patients undergoing intracytoplasmic sperm injection (ICSI) treatment at the reproductive medicine center of Angers hospital (France), between January 2014 and March 2018. Couples were referred for ICSI because of male factor infertility, prior fertilization failure, or endometriosis. Indeed, at our center, we perform ICSI for patients with confirmed endometriosis, in order to optimize fertilization ,17. The In our center, the indications for endometriosis surgery before or after Assisted Reproductive Technology (ART) in case of endometrioma (OMA) or deep infiltrating endometriosis (DIE) respect the French recommendations published in 2018 and inspired by the recent literature on the subject . The benThe study group included all patients with endometriosis, the control group included all other non-endometriosis patients. All patients had a baseline fertility assessment prior to IVF that included basal day 3 FSH and anti-Müllerian hormone (AMH) level, as well as a pelvic ultrasound for Antral Follicle Count (AFC). Cycles were performed routinely without any additional intervention required by this study. All participants did not object to the processing of their personal data.®, Merck, Lyon, France) when at least 4 follicles reached a size greater than 17 mm. Oocyte retrieval was scheduled 36 h after the hCG injection and performed under general anesthesia.Patients were stimulated either with a long gonadotropin-releasing hormone (GnRH) agonist or an antagonist protocol. At our center, we offer the long agonist protocol first line for patients with endometriosis, based on studies showing better pregnancy rates . For the®  and denuded one hour after, using hyaluronidase  and mechanical stripping. Sperm preparation was achieved by a 40/80% discontinuous density gradient using PureSperm® , or by washing with FertiCult® IVF medium  in cases of cryptozoospermia. Metaphase II (MII) oocytes, i.e., oocytes that expelled the first polar body 1 h after oocyte collection, were injected with spermatozoa for the ICSI procedure. Fertilization was assessed 18 h post-injection. Embryos were cultured in Global medium®  with 10% of Human Serum Albumin  at 37 °C under an atmosphere containing 6% CO2 and 7% oxygen and were conventionally observed, once every day, until the transfer or the cryopreservation. We used the ESHRE classification system to assess embryo morphology at the cleavage stage and Gardner’s classification at the blastocyst stage [The cumulus-oocyte complexes were washed in multiple dishes with Flushing mediumst stage . Only thEmbryo transfer was generally performed on day 2 or day 3 after the oocyte retrieval for a first or second cycle and otherwise on day 5. Only one or two embryos were transferred, depending on the patient age, the cycle rank, and the embryo quality, but irrespective of the endometriosis status of the patient. Overall, for the first transfer in women under 35 years old, we transferred only one embryo if embryo freezing was possible. For women over 35, or for younger women undergoing attempts ranked above one, we transferred two embryos. Vaginal micronized progesterone (600 mg/day) was administered for luteal phase support from the day of oocyte retrieval and continued till twelve weeks gestational age (GA). Biological pregnancy was initially diagnosed by a serum bhCG level above 100 IU/L, which was tested 14 days after oocyte retrieval. A vaginal ultrasound was performed at 7 weeks GA to confirm a clinical pregnancy .All remaining good quality embryos were cryopreserved. Cleavage stage embryos were cryopreserved by slow freezing  prior to September 2016 and by vitrification  afterwards, while blastocysts were always cryopreserved by vitrification (Irvine), according to the manufacturer’s recommendations. Five hundred eighty-one frozen embryos transfers were performed, mostly substituted cycles (>90% cycles). Natural or stimulated cycles could be used in cases of recurrent implantation failure.Our primary outcome measures were the number of oocytes, the maturity rate , the number of embryos, the cleavage rate (number of day-3 embryos divided by number of MII oocytes), the proportion of top-quality embryos (proportion of embryos with the highest score on day 2—grade 1 according to the ESHRE consensus—over to the number of embryos obtained), the usable embryo yield (proportion of embryos either transferred or cryopreserved), and the number of frozen embryos. The secondary outcome measures were the implantation rate , the live births rate (delivery after 22 weeks GA) and the cumulative live births rate (using fresh and frozen embryos of different cycles for a same patient).2 test, while continuous variables were compared using the non-parametric Mann–Whitney U-test. All the calculations were computed using R [p < 0.05 (*).Categorical variables were compared using χ using R . Because using R . DiffereThe study was conducted according to the ethical standards of the Helsinki Declaration and its later amendments and with the approval of the Ethics Committee of the Angers University Hospital, France (Number 2020/66). All the data were anonymously collected from the local database, in accordance with the French National Commission for Information and Liberties (ar20-0029v0).p = 0.03). As expected, the total motile sperm count was significantly higher in the endometriosis group  because of the inclusion of male factor infertility in the control group. Mean age and tobacco use, known as confounder of ovarian reserve markers, were comparable between the two groups. Patients in the endometriosis group had a significantly lower ovarian reserve, with a significantly lower mean serum AMH level  and a significantly lower mean AFC (16 ± 10 vs 20 ± 11 p < 0.0001). However, day 3 serum FSH levels were comparable between the two groups (p = 0.1).Among the 1124 cycles reported, 155 (13.8%) ICSI cycles concerned 84 patients with endometriosis and 969 cycles (86.2%) referred to 590 patients in the control group. Baseline characteristics are described in p < 0.0001). As expected, the long agonist protocol was more often prescribed in the endometriosis group than in the control group . The type of gonadotropins used (FSH vs FSH + LH) did not differ between the 2 groups (p = 0.5) (Patients in the endometriosis group required significantly more FSH (2819 ± 1109 IU) during stimulation than in the control group (2342 ± 986 IU) (p = 0.5) .p < 0.0001) and MII oocytes  compared to the control group, but the oocyte maturity rate was comparable between the two groups . The total number of embryos  and top-quality embryos  was significantly lower in the endometriosis group compared to the control group, but there were no differences in the cleavage rate. The mean number of embryos with late cleavage (cleavage beginning on day 3) and the mean number of transferred embryos did not differ between the two groups. We found a trend towards a lower proportion of top-quality embryos in the endometriosis group, but the difference did not reach clinical significance (p = 0.06), while the proportion of usable embryos was comparable between the two groups (p = 0.1). A multivariate Poisson regression was performed to adjust for baseline differences in BMI, AMH, AFC, protocol, total dose of FSH required, and number of oocytes retrieved. The analysis did not reveal any significant association between endometriosis and the proportion of top-quality embryos ; p = 0.3).The IVF parameters are summarized in p = 0.05), the implantation rate (17.3% vs. 23.6%) and the live birth rate per cycle (16.1% vs. 23.2%) were comparable between the endometriosis and the control groups. Patients in the endometriosis group had a significantly lower number of frozen embryos , frozen embryo transfers  and thus had a significantly lower cumulative live birth rate  compared to the control group  to patients with stage III or greater endometriosis  (p < 0.0001). All the IVF cycle characteristics and outcomes were comparable between the two groups. Pelvic adhesions were described in 23% of all patients, but the description of types and degree of adhesions was not always available.In order to further investigate the potential impact of the endometriosis stage on IVF outcomes, we performed a subgroup analysis in the endometriosis group and compared patients with stage I–II ( cycles) . The patn = 90). When compared to the control group, these patients had significantly lower ovarian reserve parameters , required significantly higher doses of gonadotropins, and had a significantly lower number of oocytes retrieved and embryos. However, the implantation and live birth rates were comparable with the control group  compared to the control group. Moreover, we identified a subgroup of women with confirmed endometriosis but without any previous surgery who also had a significantly lower ovarian reserve compared to the control group. This is in accordance with most studies that show a decreased ovarian reserve in patients with endometriosis, regardless of whether they had any previous ovarian surgery ,54. EndoDespite having a significant decrease in the quantitative IVF parameters assessed, we did not find any detrimental impact of endometriosis on oocyte maturity rate and cleavage rate, and multivariate analysis did not show any association between endometriosis and embryo quality. Our findings on the embryo quality are in accordance with several reports ,63,64,65We found a trend for decreased implantation and live birth rates in women with endometriosis, but the difference was not statistically significant. However, due to the lower number of frozen embryos in patients, the cumulative live birth rates were significantly lower in the endometriosis group. The impact of endometriosis on pregnancy and live birth rates following IVF remains controversial, with some studies confirming a significant negative impact and others reporting no effect ,11,72,73Finally, our study failed to show any significant difference in embryo quality between the two groups. However, some values were close to the significance threshold and should therefore be interpreted with caution, especially since they may be subject to limits and biases. Indeed, we assessed embryo quality at the cleavage stage and were not able to assess the percentage of blastulation and blastocyst quality. Moreover, higher semen parameters  in the endometriosis group could have counteracted the potential effect of endometriosis on embryo quality, as a negative relationship between semen quality and embryo development is described in ICSI ,75. On tIn conclusion, our study showed a significantly lower number of oocytes and embryos in women with endometriosis compared to patients with other causes of infertility but found no significantly difference in the embryo quality. The live birth rates per cycle were comparable between the two groups, but the cumulative live birth rate was significantly lower in women with endometriosis as a consequence of the lower number of frozen embryos. Endometriosis remains a complex disease that impairs quality of life ,2,3 and


In this multicenter cohort study, a total of 42,231 adult nephrology outpatients from 2001 to 2016 were included. End-stage renal disease (ESRD) patients on dialysis within 3 months of the first visit were excluded. Instrumental variable (IV) was used to define regions based on the proportion of patients with serum TCO2 < 22 mEq/L. The crude mortality rate was 12.2% during a median 77.0-month follow-up period. The Cox-proportional hazard regression model adjusted for initial kidney function, alkali supplementation, and the use of diuretics demonstrated that low TCO2 concentration was not associated with progression to ESRD, but significantly increased the risk of death. The IV analysis also confirmed a significant association between initial TCO2 concentration and mortality . This result was consistently significant regardless of the underlying renal function. In conclusion, low TCO2 levels are significantly associated with mortality but not with progression to ESRD in patients with ambulatory care.Large-scale studies have not been conducted to assess whether serum hypobicarbonatemia increases the risk for kidney function deterioration and mortality among East-Asians. We aimed to determine the association between serum total CO Previous epidemiologic cohort studies showed that CKD patients with higher serum bicarbonate levels had lower risks of developing end-stage renal disease (ESRD) in various populations6. Several observational studies involving patients with CKD found that lower total serum CO2 (TCO2) levels, higher net endogenous acid production7, and high dietary acid loads9 were associated with higher risks of progressive renal function deterioration among various ethnic groups11. Reaven et al. recently conducted a well-designed observational study which included the medical records of > 50,000 community-based patients with advanced CKD (stages 3a-5) obtained from a diverse geographically distributed database12. The longitudinal study analyzed the10-year data of this large US community-based population to assess the effects of hypobicarbonatemia on mortality and kidney outcomes. The authors found that death, ESRD with ongoing dialysis, and eGFR reduction of ≥ 40% were significantly higher among patients with hypobicarbonatemia compared to those with normal serum bicarbonate levels 12. The prevalence of early CKD is increasing in South Korea, and data on the role of asymptomatic metabolic acidosis in CKD progression is limited15.Serum hypobicarbonatemia was observed in about 20% of patients with chronic kidney disease (CKD) stages 4–5 in the NHANES cohort2 on venous sampling. We attempted to determine the effects of hypobicarbonatemia in a retrospective cohort from the patients with various regions of South Korea, using instrumental variable (IV) analysis.Notably, it is difficult to evaluate acid-base status in an outpatient setting because of the difficulty associated with performing arterial blood gas analysis in this setting. In this study, we investigated the long-term clinical impact of hypobicarbonatemia on the development of incident ESRD and overall mortality in East-Asians, using the level of TCOth International Code of Disease Classification (ICD-10) was used. Data were collected using electronic health records following the methods also described in previous studies16. In detail, the date of birth, sex, age, body mass index (kg/m2), and data related to other comorbidities (using the diagnostic code by ICD-10) and medication were recorded.This was a retrospective observational cohort study of patients tracked in the outpatient nephrology clinic of two tertiary hospitals in South Korea . Hospital administrative data were extracted, and the 102 (TCO2) , patients with no address (n = 27), and those with extreme values of TCO2 (n = 72) were also excluded. The final analysis was performed including 42,231 patients  blockades, including angiotensin-converting-enzyme inhibitors  and angiotensin II receptor blockers (ARBs), and diuretics were identified. Diuretics group included acetazolamide, spironolactone, amiloride, triamterene, furosemide, torasemide, thiazide, indapamide and metolazone.The Low TCOWe included only those patients who had undergone treatment for more than 30 days in the treatment group. The CKD grade was determined by calculating eGFR from creatinine level using Modification of Diet in Renal Disease (MDRD) GFR. The study was approved by the Seoul National University Boramae Medical Center Institutional Review Board (No. 10-2018-10/031), and requirement for consent was waived by virtue of the retrospective design. All clinical studies were conducted in accordance with the guidelines of the Declaration of Helsinki as amended in 2013.2 level, and the secondary outcome was to find a new development of ESRD that required dialysis and progression to mortality from various causes. We obtained mortality information from the national statistics office17, and ESRD incidence was defined as follows: patients recently undergoing hemodialysis were extracted from the electronic medical records using the clinical data warehouse in our study centers. Patients who had started hemodialysis therapy were identified by the combination of operational definitions as follows: the occurrence of a new claim for the payment code of hemodialysis by screening ICD-10 codes, and vascular access operations; incident dialysis was also identified by the first date of when dialysis was suggested.The primary outcome was to clarify the all-cause mortality according to the TCO2 group and control groups. We included covariates, such as TCO2 values, age, sex, history of hypertension, diabetes mellitus, MDRD-GFR, and SB usage, in the final model using multivariate Cox regression analysis.We used the chi-squared test for categorical variables and the Student’s t-test for continuous variables to compare the demographics. Categorical variables were reported as the percentage of all patients, and continuous variables as the mean ± standard deviation. The multivariate Cox proportional hazards model was used to calculate the hazard ratio (HR) with a 95% CI for all-cause mortality and renal outcomes. Kaplan–Meier curves and the multivariate Cox proportional hazard models were used to compare the outcomes between the Low TCO21. The IV in this study was regional classification of proportion of patients with TCO2 < 22 mEq/L. We used the IV to independently associate the effects of low TCO2 level and clinical outcomes. All statistical analyses were performed using R version 3.5.022 (Comprehensive R Archive Network: http://cran.r-project.org) and SPSS version 22.0 . The maps in supplementary figures was generated that outlines of administrative districts were obtained from Statistical Geographic Information Service of Statistics Korea . Maps were produced with R in conjunction with the software packages ggmap, maptools, rgdal, abd ggplot226. In all analyses, P < 0.05 was considered statistically significant.To minimize the bias of this retrospective observational study, we conducted an IV analysis2 and the TCO2 was 26.2 mEq/L. CKD stage 2 accounted for 44.8% of all patients, and about 10% of patients were in advanced stage with CKD stage 4–5. As CKD progressed, a decrease in TCO2 levels was observed, and the use of sodium bicarbonate, RAAS blockade, or diuretics was more frequent.The baseline characteristics and demographics of the study population divided according to the CKD stage are shown in Table 2 level was < 22 mEq/L in 10.1% of all patients, and we graphically depicted the proportion of patients with a TCO2 < 22 mEq/L by the patient’s residential location .Meanwhile, the serum TCO2 < 22 mEq/L had a higher risk of mortality  than those in the other groups. The Cox-proportional hazard regression model adjusted for sex, age, hypertension, diabetes mellitus, and alkali supplementation showed that low TCO2 concentration increased the risk for mortality . These results were consistently significant even when the use of RAAS blockades and diuretics were additionally adjusted (model 6). In the IV analysis, low TCO2 concentration also increased the mortality risk. When adjusted for baseline renal function, alkali supplementation, RAAS blockade, and diuretics, the mortality risk reduced by approximately 44% with each 1 mEq/L increase in serum TCO2 concentration .During the median 77.0-month follow-up period, the crude mortality rate was 12.2%. According to the Kaplan–Meier curve  than the other groups in univariate analysis .The risk of progression to ESRD steadily increased over time and occurred in 8.5% of patients during the follow-up period. Patients with TCO2 level and ESRD progression risk by TCO2 level groups is also presented in Supplementary Information Fig. 2 concentration did not affect ESRD progression, regardless of eGFR  by the residence area of participants as the IV to determine the independent relationship between TCO2 level and clinical outcomes. To be selected as an IV, several important assumptions must be met20. First, the exposure values should have definite predictive associations with the causal inference for IV. This IV should not be directly related to the outcome to be predicted. The four groups in Supplementary Information Table 2 level.This study is the first population-scale study to show that low TCO2 concentrations were protective in patients at risk of all-cause mortality, but not for preventing progression to ESRD  may not indicate metabolic acidosis, and this is a possible limitation of the study. The identification of acid–base status based on TCO2 as bicarbonate level may have led to misclassification because low TCO2 might be due to chronic respiratory or mixed acid–base disorders. These misclassifications could be seen in patients with ESRD receiving hemodialysis29 and in patients with acute renal failure requiring continuous renal replacement therapy30. However, in patients with preserved renal function in an outpatient setting, TCO2 is considered effective for predicting serum bicarbonate level31. Thus, we believe that the low TCO2 group in this study mainly reflects hypobicarbonatemia, irrespective of pH status. A recent study by Hirai et al. showed that serum TCO2 correlated with bicarbonate concentration in Japanese patients with pre-dialysis CKD 32, supporting our results.Final Cox regression models and IV analysis including covariates of initial eGFR and information on the use of medications such as SB, RAAS blockade, and diuretics, showed that higher TCOD Tables . Serum b2 disappeared when baseline eGFR was considered , the influence on the ESRD of various factors other than TCO2 level itself is much greater . In the results of our study, the final Cox model and IV analysis were inconclusive in terms of ESRD incidence as there were limitations due to the retrospective study design, and heterogeneity of the operational definition of ESRD was also limitation. Another possible rationale for hypobicarbonatemia being associated only with mortality but not with ESRD incidence in our study involves systemic compensation capacity. A recent study in patients with CKD found that hypobicarbonatemia was involved in CKD progression only when pH reached acidemia (pH < 7.32)33. The acid–base status  on the clinical outcome of hypobicarbonatemia was not considered in existing studies11. According to researchers at the University of Osaka, in a study of 1,058 patients with CKD (eGFR < 60 mL/min), TCO2 level and pH were measured simultaneously through venous blood gas analysis (VBGA). This study showed that the strength of this association is significantly changed by acidemia. Hypobicarbonatemia is associated with an increased risk of ESRD only when pH is low (pH < 7.32) but not when pH is normal. This association suggests that the compensatory reservoir might be important in terms of CKD prognosis15. In our study, patients with CKD 1,2 (eGFR > 60 mL/min) were 67% of the total. These patients are likely to have a normal pH. Results from the Osaka researchers also found that 40% of the group with bicarbonate levels under 21.5 mEq/L did not present acidemia as a result of systemic compensation33. In addition, in a prospective cohort study of an elderly population without CKD, similar to our results, 60% of the group with low bicarbonate levels did not have acidemia34. This study showed a correlation between low bicarbonatemia levels and high mortality, regardless of pH, and the modification of the effects by pH did not affect this association, which supports our findings. These findings suggest that combining information on pH with bicarbonate levels, especially to improve renal outcome, helps identify patients at high risk of ESRD. In addition, needless alkali treatments could be reduced in non-acidic hypobicarbonatemic patients 33. Further studies may identify the need for clinicians to further improve non-acidicidc hypobicarbonatemic or eubicarbonatic H+ retension in early CKD patients for slowing CKD progression. Treatment and consequent improvement of acidosis in CKD patients are expected to improve the clinical outcomes; however, the optimal interventional methods and population remain inconclusive, and The cautions is needen for the replacement of alkali using SB which is associated with complications, such as deterioration of sodium retention, edema, and heart failure aggravation.In terms of ESRD development, there was significance in model 3 without eGFR as a covariate; however, the protective effect of high TCOve Table , and in 2 levels were significantly associated with mortality, but not with progression to ESRD in IV analysis using regional classification by proportion of low TCO2 as an IV. Further studies, for a prospective interventional study on a nation-wide scale for the effects of alkali therapy on CKD progression in patients with eubicarbonatemia with early CKD and explanations for possible biologic mechanisms, in Korean CKD patients are needed.In conclusion, low TCOSupplementary Information.


Panthera tigris) worldwide and all subspecies of tigers are globally endangered. Due to pressures from poaching and retaliatory killings, this carnivore had lost an estimated 93% of its historic range. Given the critical situation of these wild felines, the health of each individual is of prime importance and laboratory blood testing, as well as evaluation of their physical condition, is important in their health assessment. Protein concentrations in the blood can be altered by malnutrition and dehydration as well as by disease. Serum electrophoresis allows the identification of the different protein fractions present in the blood and represents a useful tool in the diagnosis and monitoring of a number of diseases. Due to the nature of wild Panthera tigris, it is extremely difficult to obtain biological samples from free-living subjects, and therefore the values obtained from captive tigers provide very useful data. This study reports serum protein electrophoresis in 11 adult captive individuals. These results will be useful for the evaluation of physiological and pathological alterations in wild and captive tigers and populations.The tiger is the largest of the wild cats. There are fewer than 4000 wild tigers (Panthera tigris), the health of each individual is important and any data on blood chemistry values can provide valuable information alongside the assessment of physical condition. The nature of tigers in the wild makes it is extremely difficult to obtain biological samples from free-living subjects, therefore the values obtained from captive tigers provide very useful data. Serum protein electrophoresis is a useful tool in the diagnosis and monitoring of a number of diseases. In this study, we evaluated agarose gel serum protein electrophoresis on samples from 11 healthy captive tigers. Serum electrophoresis on all 11 tiger samples successfully separated proteins into albumin, α1, α2, β1, β2 and γ globulin fractions as in other mammals. Electrophoretic patterns were comparable in all tigers. Mean± standard deviation or median and range values obtained for each protein fraction in healthy tigers were, respectively: 3.6 ± 0.2, 0.21 (0.2–0.23), 1.2 ± 0.2, 10.7 ± 0.2, 0.4 (0.3–0.6), 1.2 (1–1.8) gr/dL. The results of this preliminary study provide the first data on serum electrophoretic patterns in tigers and may be a useful diagnostic tool in the health assessment of this endangered species.Given the endangered status of tigers ( Panthera tigris) are left in the world [Panthera tigris) as globally endangered [The tiger is the largest of the wild cats . This lahe world  and the dangered .Panthera tigris altaica). Today, there are fewer than 400 Amur tigers left in Russia and the eastern region of northeastern China [Panthera tigris tigris) is the most numerous of all tiger subspecies, with a wild population of more than 2500 [Approximately 10% of the world’s tigers are found in the Russian Far East, where a single metapopulation represents the vast majority of Siberian, or Amur, tigers , resulting in a typical electrophoretic pattern for the distribution of proteins [1 and α2, globulin fractions, are a group of serum globulins, including ceruloplasmin, haptoglobin, a-2-macroglobulin, alpha1-acid-glycoprotein, that increase during acute inflammation, infection, surgical trauma or stress [Given the critical situation of these wild felines, maintaining the health of each individual is essential and any hematological and blood chemistry values that can provide information on the nutritional health status and physical condition are valuable. Furthermore, the detection of signs of disease in these animals is difficult and biological parameters are regarded as very useful complementary tools in the diagnosis and treatment of possible pathologies . Total pproteins . Serum pproteins . In veteproteins . In domeproteins . Wild caproteins . Hypergaproteins ,20 and lproteins . Moreoveproteins , evaluatproteins  to ensurproteins .The acutr stress . It has r stress . Panthera tigris means it is extremely difficult to obtain biological samples from free-living subjects, making the data derived from captive individuals all the more useful. In addition, access to large numbers of free-living or captive individuals for testing is limited and researchers rely on data from a limited number of individuals [The wild nature of ividuals ,25.Panthera tigris. Such data are valuable for the veterinary care of these animals. The objective of this study was to report values of total serum protein and electrophoresis fractions for healthy captive Panthera tigris, belonging to the subspecies Panthera tigris tigris  and Panthera tigris altaica (Siberian tiger). These results will be useful for the evaluation of physiological and pathological alterations in wild and captive tiger individuals and populations.To the knowledge of the authors, there is a paucity of literature regarding the serum electrophoretic pattern of Panthera tigris tigris), 7 tigers (Panthera tigris) and 2 Siberian tigers (Panthera tigris Altaica), including 7 neutered males and 8 neutered females, with ages ranging from 3.5 to 17 years. All tigers were housed, individually or in groups based on individual sociability, in various sized enclosures, rescue centers for exotic felids, zoological parks, or a circus located in northern Italy. Tigers were being immobilized for routine physical examination, ocular and dental examination, vaccination administration or minor surgical or diagnostic procedures. Each tiger received a clinical examination, and those with any visible abnormalities, with an inadequate body condition score, with signs of dehydration or with any signs of disease were excluded from the group of subjects considered clinically healthy. Sera were collected from fifteen tigers, 6 Bengal tigers  and serum was obtained by centrifugation at 10 min at 2500× g. Written owner consent for use of surplus blood samples, and use of data for scientific purposes, was obtained during consultations. The study design was approved by University of Milan Animal Welfare Bioethical Committee . As part of the health examination while under general anesthesia, blood samples were collected from the jugular, cephalic or saphenous vein of each animal. Blood was collected in plain tubes  on a Cobas Mira Classics Roche automated chemistry analyzer .1-β2 . Serum was electrophoresed for 7 minutes at 33 volts hours and stained with diluted Amidoschwarz dye at pH 2 (4 g/L Amidoschwarz dye and 6.7% ethylene glycol). The AGE procedure was conducted according to the manufacturer’s instructions, and commercial human serum was used as the control . Using the computer software Phoresis for Windows 2000 or XP Pro (Sebia), the electrophoretic curve for each sample was displayed. Protein fractions were determined as the percentage optical absorbance, and the absolute concentration in g/dL was automatically calculated from the total serum protein concentration. Albumin to globulin (A/G) ratios were also calculated. The same operator analyzed all samples. To establish the inter-assay-accuracy of agarose gel electrophoresis on tiger serum, sera from two healthy tigers, one Bengal Tiger, 9A, F and one Siberian Tiger 1,1A, F, were tested 3 times on the same day, in the same laboratory and interpreted in duplicate by two operators. Coefficient of variability (CV) computed as SD/mean × 100 was calculated for each protein fraction.Sera samples were refrigerated at 4 °C and were analyzed within 8 hours of sampling. Protein fractions were analyzed using a semiautomated agarose gel electrophoresis (AGE) system , five tigers (Panthera tigris) and one Siberian tiger (Panthera tigris Altaica), four neutered males and seven neutered females, with ages ranging from 5 to 16 years—were used to identify electrophoretic patterns of serum proteins in healthy tigers. Agarose gel electrophoresis carried out on all 11 tiger samples, successfully separated tiger serum proteins into albumin, α1, α2, β1, β2 and γ globulin fractions  serum protein electrophoretic fractions by capillary electrophoresis (CE). Although the AGE and CE are different techniques for protein fraction separation, the shape of the electrophoretogram of cheetah serum was comparable to that found in our healthy tiger sample [Although the sample size analyzed was limited, the data obtained in this study could suggest that healthy tigers may have a higher concentration of acute phase proteins than domestic cats, or that the tiger population studied could have been in an inflammatory state. The interpretation of serum protein electrophoretic patterns depends on the variations among different groups of animals. Moreover, acute phase proteins are a variable group of serum proteins and concentrations vary widely between different animal species . Domestir sample .1 globulin fraction exceeded the acceptable value of 20.83%. This result could be due to the low concentration of α1 globulins in the serum. In fact, the accuracy of analysis is usually better for protein fractions found in higher serum concentrations because low concentrations are more susceptible to small changes [The percentage of variation observed between protein fractions of two healthy tiger serum samples repeatedly submitted to agarose gel electrophoresis to evaluate inter-assay-accuracy were within the accepted ranges of within-subject biological variation for people . Only th changes . Cushing changes  describeThere were a number of limitations with this study. Firstly, although each tiger was clinically examined, and screened for visible alterations and low body condition score, the history was sometimes incomplete or unavailable, which may have compromised the accurate categorization of animals according to health status and disease type. In addition, samples were from animals in different types of housing, so the diversity of habitat and diet could have affected the results. . FurtherIn veterinary medicine, serum protein electrophoresis is recognized as a useful tool in the diagnosis, prognosis and monitoring of a number of diseases ,17. AltePanthera tigris, it is extremely difficult to obtain biological samples from free-living subjects, and therefore the values obtained from captive tigers provide very useful data. Results indicate that agarose gel electrophoresis separates the total serum protein into six fractions, albumin, α1, α2, β1, β2 and γ globulins in tigers as in other mammals, resulting in a typical electrophoretic pattern for the distribution of proteins. Mean values of albumin and globulin fractions, with the exception of α2 and β1 globulins mean concentration, in our healthy tiger population fell within the reference values indicated for protein serum electrophoresis performed with AGE in domestic cats. These preliminary results provide the first data on serum electrophoretic pattern in healthy tigers and may offer a platform for further research into serum proteins as a useful diagnostic tool in the health assessment of this endangered species. Serum protein electrophoresis is a useful tool in the diagnosis and monitoring of a number of diseases. This study presents serum protein electrophoresis in a sample of healthy captive tigers. Due to the nature of wild