Neisseria gonorrhoeae, the causative agent for sexually transmitted infection (STI) gonorrhoea, has emerged with a significant public health impact on acquiring resistance to antimicrobials available for treatment. The resistance of N. gonorrhoeae limit treatment options and contributed to high morbidity associated with gonorrhoea. Data on antimicrobial resistance (AMR) profiles in N. gonorrhoeae is scares in Zambia. This study aimed to determine the antibiotic susceptibilities in N. gonorrhoeae isolates from Lusaka, Zambia.2 for 24 h. Identification of N. gonorrhoeae isolates was achieved by Gram stain, oxidase, nitrocefin disk, BactiCard Neisseria, and Viteck® Compact. The AMR profiles were determined using E-test. Statistical significant was determined by Pearson’s Chi-square test, Mann-Whitney U test, or logistic regression with p-values of < 0.05 indicating significance.A prospective cross-sectional study was conducted on 630 STI patients who presented with urethral or vaginal discharge from 2019 to 2020. Urethral and endocervical secretions were cultured on Modified Thayer Martin agar and incubated at 36 °C ± 1 °C in 5% CONeisseria gonorrhoeae was isolated from 19.4% (122/630) patients of which 72.9% (89/122) were male, with highest prevalence of isolation in the age category of 25–34 years. The prevalence of resistance was high to penicillin (85.2%), tetracycline (68.9%) and ciprofloxacin (59.8%) with MIC90 of 32 µg/mL, 8 µg/mL, and 8 µg/mL respectively. The isolates had reduced susceptibility to cefixime (1.6%), spectinomycin (4.9%) and (4.9%) for azithromycin. All isolates were susceptible to ceftriaxone. Risk factors associated with AMR were douching in females , female gender , HIV-positivity , no condom use or unprotected sex , sex trading , and over-counter treatment of ciprofloxacin .A total of 630 patients were recruited of which 46% (290/630) with the median of 29 years and interquartile range (IQR) of 19–39 years were male. The median of the females was 26 years and IQR of 15–37 years. N. gonorrhoeae resistance to penicillin, tetracycline and ciprofloxacin was high necessitating revision of the treatment guidelines. However, no resistance to ceftriaxone was detected. Therefore, monitoring of antibiotic resistance remains critical in Zambia.The The online version contains supplementary material available at 10.1186/s12879-022-07674-y. Neisseria gonorrhoeae, which remains a major global public health concern because of its capacity to acquire high levels of resistance to antimicrobial agents available for treatment [Neisseria gonorrhoeae has dramatically developed plasmid mediated and/or chromosomally mediated antimicrobial resistance (AMR) leading to the removal of the antimicrobial agent usages as standard first line treatment regimens [Gonorrhoea is a sexually transmitted infection (STI) caused by the bacterium reatment , 2. In tregimens . The emeregimens –6.Chlamydia trachomatis infections among adults of 15–49 years of age [In 2020, the World Health Organization (WHO) estimated 82.4 million incident global cases of gonorrheoa, and second commonly sexually transmitted disease after s of age , 8. The s of age –12.Neisseria gonorrhoeae infects the genital tract in women and men and can also be transmitted from mother to child during delivery and cause infection of the eye of the newborn [N. gonorrhoeae to extra-genital sites causes endocarditis, septic arthritis and meningitis [ newborn –16. Comp newborn , 17–28. ningitis , 30. In ningitis –35.N. gonorrhoeae for proper selection and use of antimicrobial agents despite resistance been an emerging phenomenon in Zambia. Multidrug-resistant (MDR) N. gonorrhoeae was defined as isolates resistant to either 3GCs or azithromycin, plus at least two antimicrobial agents , and extensively drug-resistant (XDR) N. gonorrhoeae been resistant to 3GCs and azithromycin, plus at least two antimicrobial agents  [The syndromic case management of sexually transmitted infections (STIs) which had contributed highly to AMR due to empirical treatment was being used in many countries in sub-Sahara Africa , 36, 37.cycline) .Neisseria gonorrhoeae isolated from patients attending sexually transmitted infection clinics in urban hospitals, Lusaka, Zambia.The aim of this study was to determine the antimicrobial resistance profile of Neisseria gonorrhoeae isolated from 630 urogenital specimens from patients attending STIs clinics in urban hospitals in Lusaka, Zambia. The urethral and endocervical specimens were collected from patients who presented with a discharge from September, 2019 to August, 2020, and submitted for testing to microbiology laboratory at the University Teaching Hospital. The UTH microbiology laboratory participates in WHO and Southern Africa Development Accreditation Service (SADCAS) programs.A prospective cross-sectional study on 122 The data was collected by trained nurses, clinical officers and medical doctors through face to face interviews technique using structured questionnaires to collect demographical and clinical factors from consented study participants. The questionnaires were adopted after reviewing different research studies done so far in the region. Urethral swabs from male participates were collected by inserted a flexible wire 2–3 cm into the urethra and rotated gently before withdrawing. In female participants the speculum was inserted to visualise the cervix and a Dacron swab on a rigid shaft was inserted 2–3 cm into the endocervix and removed with rotation from the endocervical canal. The swabs were immediately put in Amies transport media and transported to UTH microbiology laboratory for testing within 15 min of collection.2 for 24 h, and then the plate was read for growth. Identification of the isolates was achieved by Gram stain , oxidase strips , BactiCard Neisseria kit , and Vitek® Compact using NH ID cards  according to the manufacturer’s protocol. The β-lactamase disk (Mast Group Ltd. Merseyside. UK. Ref. D59) was used to identify penicillinase-producing Neisseria gonorrhoeae strains (PPNG), and isolates were regarded as having a high level of resistance to tetracycline (TRNG) with MIC values ≥ 16 µg/mL.Urethral and endocervical secretions were inoculated on Modified Thayer Martin medium and incubated at 36 ºC ± 1 °C in 5% COoC ± 1o C in 5% CO2 for 24 h. The SIR categories for antimicrobial agents in µg/mL were as follows: Ciprofloxacin (CIP) S; ≤0.06, I; 0.12–0.5, R; ≥1, ceftriaxone (CTX) S; ≤ 0.25, R; > 0.25, spectinomycin (SPEC) S; ≤ 32, I; 64, R ≥ 128, cefixime (CFX) S; ≤0.25, R; >0.25, azithromycin (AZT) S; ≤1, R; >1, penicillin (PEN) S; ≤0.06, I; 0.12-1, R; ≥2, and tetracycline (TET) S; ≤0.25, I; 0.12-1, R; ≥2. Neisseria gonorrhoeae American Type Culture Collection (ATCC) 49,226 was used as a reference strain and was within the acceptable quality control ranges.The minimum inhibitory concentrations  of ciprofloxacin, ceftriaxone, spectinomycin, azithromycin, penicillin, and tetracycline were determined by E-test , on GC-chocolate with 1% Vitox supplement  following the manufacturer’s instructions. The interpretation of MIC dilutions in susceptible (S), intermediate (I) and resistance (R) categories were according to Clinical and Laboratory Standard Institute (CLSI) criteria . The plap-values and 95% confidence interval were computed. A p-value of < 0.05 was taken as indication of statistical significance.Raw data was cleaned, audited and exported to Stata statistical software version 12.1  for analysis. Descriptive statistics was reported as frequencies and proportions. The Pearson’s Chi-square test was used to determine the bivariate association between individual categorical variables and the outcome variable. Mann-Whitney U test was used to determine the significant difference in the median age of study participants, and median and interquartile ranges were computed. Univariate and multiple logistic regression was used to assess the association between outcome variable and independent variables; the odds ratio (OR), Neisseria gonorrhoeae was isolated from 19.4% (122/630) of which 72.9% (89/122) were from male. The gonococcal (GC) infection was much higher in males accounting for (73%), and (54.9%) of the participants with GC infection were married. The highest prevalence of GC infection (47.5%) occurred in the age group of 25–34 years, participants without tertiary education (73.7%), employees (68.9%), men who were circumcised (53.9%), and among men (50.8%) of who used condom during sexual intercourse. The HIV positivity among the participants was (26.2%), traded sex (48.8%), over counter treatment (27%) and (36.4%) of females participants were douching. The majority of GC infection (36.9%) were reported from University Teaching Hospital which was the largest and main referral medical centre in Zambia  were female. p = 0.092 , and the median for females was 26 years . There was no significant difference in median age between males and females across the categories of gender, N. gonorrhoeae isolates, and 85.2% (104/122) isolates were resistant to penicillin with MIC values ranging from 0.047 to 32 µg/mL, MIC50 of 32 µg/mL and MIC90 of 32 µg/mL, while 68.9% (84/122) isolates were resistant to tetracycline with MIC range of 0.032 to 64 µg/mL, MIC50 of 4 µg/mL and MIC90 of 8 µg/mL. For ciprofloxacin, 59.8% (73/122) isolates were resistant with MIC values ranging from 0.016 to 32 µg/mL, MIC50 of 2 µg/mL and MIC90 of 8 µg/mL. Only 1.6% (2/122) isolates were resistant to cefixime with MIC range of 0.016 to 1 µg/mL, MIC50 of 0.032 µg/mL and MIC90 of 0.19 µg/mL. The resistance of isolates to azithromycin was 4.9% (6/122) with MIC ranging from 0.016 to 4 µg/mL, MIC50 of 0.125 µg/mL, and MIC90 of 0.75 µg/mL. All isolates were susceptible to ceftriaxone. The PPNG was detected in 45.1%, and TRNG in 2.5%, whereas 1.6% of the isolates presented both phenotypes (PPNG/TRNG).Table N. gonorrhoeae resistance to ciprofloxacin, tetracycline and penicillin were gender; female , HIV-positivity , no condom use or unprotected sex , sex trading  over-counter treatment of ciprofloxacin  and douching in females   isolates were resistant to three or more of the tested antimicrobial agents of which 4.9% (6/122) were MDR. Out of six MDR isolates, five were associated with azithromycin resistance and one isolate was associated with cefixime resistance. Only 5.7% (7/122) isolates were resistant to four antimicrobial .agents of which one isolate was XDR showing resistance to cefixime, azithromycin, ciprofloxacin and tetracycline (Fig. 2 isolateNeisseria gonorrhoeae circulating in Lusaka, Zambia are highly resistant to ciprofloxacin, penicillin and no isolate was resistant to ceftriaxone. The resistant N. gonorrhoeae was seven times more likely been isolated from older than younger patients. This could be attributed to vulnerability of the elderly to infections due to weaker immune systems subsequently intensive use of treatment drugs. The female patients are more associated with resistance than men, and those female patients who douche are six times more likely to suffer from AMR than those who were not practicing douching. Statistical association was found between douching and adverse health outcome among Zambian women [N. gonorrhoeae. These results are concordant with findings of the study done in Zimbabwe where HIV positivity, unprotected sex, and self-treatment were associated with high prevalence and resistance of N. gonorrhoeae among sexually active age groups [Gonococcal infections have been on the increase and could be attributed to different factors in different regions . Data pran women . HIV pose groups .N. gonorrhoeae showed higher levels of resistance in ciprofloxacin, penicillin and tetracycline as compared to non MDR and XDR N. gonorrhoeae. The superbugs showed resistance to cefixime and no resistance to ceftriaxone. N. gonorrhoeae strains were highly resistant to penicillin, tetracycline and ciprofloxacin. This is consistent with studies conducted in Europe, and East Asia where resistance to previously recommended treatment such as ciprofloxacin was generally above 50% [The MDR and XDR bove 50% . Accordibove 50% . The finN. gonorrhoeae associated with various demographics and clinical variables. The N. gonorrhoeae showed high resistance to ciprofloxacin, penicillin and tetracycline which poses a treatment challenge. The high level of resistance in ciprofloxacin and tetracycline highlights the need for revision of the national treatment guidelines, and monitoring of antibiotic resistance remains critical in Zambia.This study showed high prevalence of AMR of The isolates studied were only from three hospitals in Lusaka and might not be representative of other settings in Zambia.Additional file 1: TableS1. Association of demographics and clinicalvariables with N. gonorrhoeaeresistance to ciprofloxacin.Additional file 2: TableS2. Association of demographics and clinicalvariables with N. gonorrhoeaeresistance to tetracycline.Additional file 3: TableS3. Association of demographics and clinicalvariables with N. gonorrhoeaeresistance to penicillin.


To explore the potential relationship between NLR and micronutrient deficiency in patients with severe COVID-19 infection.  Sixteen patients were categorized into the mild group (mild COVID-19) and severe group (severe COVID-19) based on the guideline of the management of COVID-19. The lactate dehydrogenase (LDH); superoxide dismutase (SOD), the inflammatory markers (neutrophil lymphocyte ratio (NLR)), erythrocyte sedimentation rate (ESR), c-reactive protein (CRP), selenium (Se), iron (Fe), zinc (Zn), nickel (Ni), copper (Cu), chromium (Cr), cadmium (Cd), arsenic (As), and manganese (Mn) were measured in the blood. P < 0.05) and the level of Se (P < 0.01), Fe (P < 0.05), and Zn (P < 0.05) were significantly decreased in the severe group. The level of Se, Fe, and Zn was significantly correlated to NLR levels. Furthermore, close positive correlation was found between NLR and severity of COVID-19.  Compared to the mild group, the NLR ( The micronutrient deficiency in the blood is associated with NLR in the severity of COVID-19 patients. It has been more than two years since the outbreak of COVID-19 in Wuhan, China. Despite effective measures such as wearing masks and social distancing, the cumulative number of confirmed cases and deaths worldwide continued to increase, reaching 242 million and 4.92 million, respectively, by 21 November 2021.Research and medical management related to SARS-CoV-2 has evolved significantly in 2 years since the beginning of the epidemic. The respiratory tract, identified as the primary tissue for transmission of SARS-CoV-2, usually begins with influenza-like symptoms, but a certain percentage of patients may suffer a severe course of infection that can further lead to severe pneumonia, respiratory failure, and even death.COVID-19 cases have confirmed that respiratory viral infections often lead to inflammatory progression , 3. The Micronutrients, such as selenium and iron, are essential to keeping immune system homeostasis. Selenium is necessary for the proliferation and differentiation of the immune cells –16. The The aim of this study was to explore the potential relationship between NLR and micronutrient deficiency in critically ill patients.This study was approved by the ethics committee of Qilu Hospital of Shandong University . All the participants in this study were enrolled between January 27 and May 18, 2020. Samples for the SARS-CoV-2 test were obtained from oropharyngeal swabs or nasopharyngeal swabs in all patients following “technical guidelines for COVID-19 laboratory testing, version 2.” Pathogen examination was performed using an ORF1ab/N Gene Nucleic acid detection kit  according to manufacturer's instructions.All patients were divided into mild and severe groups based on the “Guideline on the Management of COVID-19” published by the National Health Commission of the People's Republic of China (version 8.0).3 was added for predigestion overnight. The microwave digestion instrument  was used for digestion. Acid was driven to about 0.5 mL, which was carefully transferred to a 10 mL volumetric bottle, and the digestion tank was washed with a small amount of water. Transfer the lotion to a volumetric flask and add water to the volume. Iron was diluted 500 times with water in a 10 ml volumetric flask and then measured on a machine. At the same time, the blank test of the reagent was done.An electronic balance  was used to weigh an appropriate amount of samples into the digestion tank, and 8 mL HNOCoupled plasma mass spectrometry ; working conditions: Rf power: 1350 W; sampling depth: 8.0 mm; plasma gas flow rate: 14.9 L/min; carrier gas flow: 1.2 L/min; diameter of sampling cone: 0.8 mm; diameter of the interception cone: 0.4 mm.μg/mL, lot: M2-MeB657244.Barium, lead, cadmium, arsenic, aluminum, chromium, nickel, copper, vanadium, manganese, antimony, zinc, cobalt, iron, silver, beryllium, magnesium, thallium, and other 22 kinds of mixed standard storage solution: 20 μg/L, 1.00 μg/L, 2.00 μg/L, 5.00 μg/L, 10.0 μg/L, and 20.0 μg/L, and the diluent was 1% HNO3.The Se standard solution was diluted by 22 kinds of multielement mixed standard reserve solution to the concentration of 0 μg/L, 0.500 μg/L, 1.00 μg/L, 2.00 μg/L, 5.00 μg/L, and 10.0 μg/L, and the diluent was 1% HNO3.The Ni standard solution was diluted by 22 kinds of multielement mixed standard reserve solution to the concentration of 0 μg/L, 0.500 μg/L, 1.00 μg/L, 2.00 μg/L, 5.00 μg/L, 10.0 μg/L, and 20.0 μg/L, and the diluent was 1% HNO3.The standard Mn solution was diluted by 22 kinds of multielement mixed standard reserve solution to the concentration of 0 μg/L, 10.0 μg/L, 20.0 μg/L, 50.0 μg/L, 100 μg/L, and 500 μg/L, and the diluent was 1% HNO3.Fe standard use solution was diluted by 22 kinds of multielement mixed standard reserve solution to the concentration of 0 μg/L, 5.00 μg/L, 10.0 μg/L, 20.0 μg/L, 50.0 μg/L, and 100 μg/L, and the diluent was 1% HNO3.Cu standard use solution was diluted by 22 kinds of multielement mixed standard reserve solution to the concentration of 0 μg/L, 0.100 μg/L, 0.500 μg/L, 1.00 μg/L, 2.00 μg/L, 5.00 μg/L, and 10.0 μg/L, and the diluent was 1% HNO3.The standard use solution of Cr was diluted by 22 kinds of multielement mixed standard reserve solution to the concentration of 0 μg/L, 0.100 μg/L, 0.500 μg/L, 1.00 μg/L, 2.00 μg/L, and 5.00 μg/L, and the diluent was 1% HNO3.The standard use solution of Cd and As was diluted by 22 kinds of multielement mixed standard reserve solution to the concentration of 0 t-test . The correlation analysis was done by using Statistical Package for Social Sciences (SPSS) version 28 , and Pearson's correlation analysis test was used in the analysis.Results are expressed as mean ± SEM. Normality was tested for biological data. If the data were not normally distributed, then they were log-transformed. The data were analyzed using an unpaired P=0.0419, P=0.0047), temperature (P=0.0146), national early warning score (NEWS) (P < 0.0001), lactate dehydrogenase (LDH) (P=0.0025), and superoxide dismutase (SOD) (P < 0.0001) in the severe group were significantly higher than the mild group  value in the severe group was significantly higher  , Fe (P < 0.05), and Zn (P < 0.05) had a significant correlation with NLR level after correlation analysis. Se, Fe, and Zn were all negatively correlated. There was no significant correlation between Ni, Cd, Cu, Cr, As, Mn, and NLR (The Se ( and NLR .The results of this study showed that there were significant differences in Se, Fe, Zn levels, neutrophil count, and lymphocyte count between the two groups. Compared with the mild group, the levels of selenium, iron, zinc, and other trace elements in the severe group were significantly decreased. We found that the severity of COVID-19 was positively correlated with neutrophil count and negatively correlated with lymphocytes, as confirmed by another previous study. Selenium, iron, and zinc levels were negatively correlated with NLR levels.Previous studies have reported that blood LDH levels can be used as an indicator of COVID-19 severity in large numbers of patients infected with SARS-CoV-2 in China. Higher LDH results in poorer prognosis in patients with SARS-CoV-2 infection . This isSystemic inflammation is a marker of the progression of COVID-19 and can affect multiple organ function. In severe cases, an increased systemic inflammatory response can trigger a cytokine storm, leading to organ failure and even death. CRP is a sensitive indicator of acute systemic inflammatory response after viral infection and tissue injury. In previous studies, CRP levels were found to be positively correlated with the severity of COVID. The CRP level in the severe group was significantly higher than that in the nonsevere group . We alsoLeukocytes are also involved in the systemic inflammation induced by the SARS-CoV-2 infection. Neutrophils are the most abundant white blood cells in the body and are vital to the immune system. Neutrophils tend for rapid chemotaxis to gather at the site of infection, play a defensive role, and inhibit and kill pathogenic microorganisms. A higher number of neutrophils indicates a protective effect at high levels of inflammation, but if neutrophils are too numerous, too widespread, and for too long, they can harm the lungs and cause pneumonia . This isPrevious studies have shown that micronutrient balance is essential for enhancing immune response and improving antiviral function . MicronuOur study has limitations. First, the number of local patients in this study was low, and we were unable to include more local cases due to China's excellent disease prevention and control strategy. Second, micronutrient treatments were not included in the study. Despite these factors, our study may help inspire clinicians to better understand the relationship between NLR and micronutrients.Serum selenium and iron deficiency are associated with neutrophil and lymphocyte counts and severity of COVID-19. Micronutrients should be considered in the treatment of COVID-19, especially in critically ill patients.


Our data showed that 1-MCP fumigation had a striking preservation effect on maintaining weight loss, fruit firmness, lignin, Vc content, and moisture content, inhibiting respiratory intensity and ethylene release rate, as well as decreasing cell permeability and malondialdehyde (MDA) accumulation and maintaining cell membrane integrity of root mustard. In addition, lipoxygenase (LOX), pyruvate dehydrogenase (PDH), and polyphenol oxidase (PPO) activities were significantly reduced throughout the storage period. In contrast, the activities of succinate dehydrogenase (SDH), superoxide dismutase (SOD), ascorbate peroxidase (APX), phenylalanine deaminase (PAL), and peroxidase (POD) remained at high levels. Results showed that 1-MCP treatments were effective in maintaining the quality of root mustard, and the preservation effect of 1.0 μL·L−1 1-MCP was better than other concentrations of 1-MCP. This study could serve as a theoretical reference for root mustard preservation.Root mustard is plentiful in vitamins and minerals but shrivels and molds easily. In this study, freshly harvested root mustard was fumigated with various concentrations of 1-Methycyclopropene(1-MCP) (1 µL L Brassica juncea var. megarrhiza Tsen et Lee), also known as kohlrabi and turnip, is a genus of Brassica in the cruciferous family [Root mustard  of 1-MCP , and the cover was opened for ventilation for an hour. Then, the root mustard was packed in 0.03 mm perforated polyethylene plastic bag and stored at ambient temperature (17 ± 1 °C) with a relative humidity of 65% to 75% for 35 d. The relevant indexes were measured in 7-day intervals, and the experiment was repeated three times.The root mustard was randomly divided into four groups of 85 and placed in a 60 L plastic barrel. The root mustard was sealed fumigation treatment for 24 h by using different concentrations (0 μL·L1/g. The weight loss (%) is based on the equation below (1).Weight loss rate was ascertained by the weighing approach, and initial mass was fixed for each group, recorded as m/g, and the mass measured every 7 days was recorded as m−1 [Hardness measurements were conducted at ambient temperature using a food texture analyzer (FTA) . The central part of the root mustard (1 cm × 2 cm × 1 cm) was taken and placed on a plate, then a cylindrical plunger with a diameter of 3 mm was pushed into the sample at a speed of 100 mm·min−1 ,37; 9 po2 concentration, V represents the volume of the container used for determination, and h represents measurement time.Root mustard was transferred to a 2.5 L plastisol box, allowing carbon dioxide and oxygen to accumulate in the box at 17 °C. After 2 h, gas analyzer was measured using a carbon dioxide analyzer , calculated according to Formula (2).Two root mustards were transferred to a 2.5-litre plastic box with a pinhole-sized hole on the lid for subsequent determination and sealing, allowing ethylene to accumulate in the box at 17 °C. After 24 h, gas analyzer  was connected to the injection head to measure the ethylene content.1). The solution was boiled for 30 min, allowed to cool, and then the maximum conductivity (C) was measured. The experiment was repeated 3 times and the cell membrane permeability was represented by relative conductivity. The relative conductivity is calculated according to Formula (3).Cell membrane permeability refers to the approach of Wei et al.  with som−1. The sample (1 g) was ground to homogenate with 5% trichloroacetic acid (2 mL) and quartz sand, and then ground again after adding trichloroacetic acid (8 mL). The homogenate was centrifuged at 4000 r·min−1 for 10 min, and the supernatant was collected. Add 2 ml 0.6% thiobarbituric acid solution to the supernatant and mix well. The mixture was placed in boiling water for 10 min, and then, after cooling, centrifuged at 3000 r·min−1 for 15 min. The absorbance values at 532 nm, 600 nm, and 450 nm were determined with 0.6% thiobarbituric acid solution as blank. The experiment was repeated three times. MDA content is calculated according to Equation (4).Malondialdehyde (MDA) content was assayed as described by Zhang & Liu  and was 1 is the volume of sample taken for the determination/mL; VT is the total volume of the extract/mL; W is the mass of the sample/g; 1.55 × 10−1 is the micromolar extinction coefficient of MDA.V−1. Samples of root mustard were ground into a homogenate in 2% oxalic acid solution. The final volume was 100 mL. After filtering, the 10 mL filtrate was titrated with the calibrated 2.6-dichloro indophenol sodium solution. When the color of the solution changed to pink, the dye usage was noted. Vit C content was calculated according to Equation (5).Vit C content was determined by titration of 2,6-dichlorophenol indigo  and denoV is the number of ml of dye used to titrate the sample; V1 is the milligrams of dye used in blank titration; A is 1 mL dye solution equivalent to milligrams of ascorbic acid; B is the number of milliliters of the sample solution taken during titration; b is the total milliliters of sample solution after dilution; a is the number of grams of sample.W means the 100 g sample contains mg of ascorbic acid; −1 for 20 min, repeated twice, and the supernatant was poured out. The ethanol: n-hexane = 1:2 (V:V) was extracted for 20 min, then centrifuged at 8000 r·min−1 for 20 min and repeated twice. Afterward, the precipitate was dried at ventilation for 20 h, and 25% bromoacetyl glacial acetic acid (3 mL), 2 mol·L−1 sodium hydroxide (0.9 mL), 5 mL acetic acid and 7.5 mol·L−1 hydroxylamine hydrochloride (0.1 mL) were added into the dry matter. Finally, acetic acid was fixed to 15 mL, and the absorbance values were measured at 280 nm. The lignin content was calculated according to Equation (6).The lignin content was assayed according to the approach of Zhi et al.  with somVr represents the total volume of the extract (mL), W represents sample quality (g), and Vs represents the liquid volume of enzyme used for determination (mL).Root mustard (1 g~3 g) was placed on the sample plate, the sample room was closed, and the moisture content in the sample was determined according to the instrument operation procedure.−1, 5.0 mL) and all compounds were mixed equally. The pH of the mixture was adjusted to 9.0 with hydrochloric acid, and the volume was adjusted to 500 mL with borate buffer (pH = 9.0). Crude enzyme solution: Samples (0.5 g), 20 mL phosphate buffer (pH = 7.0) and 20 mL polyvinylpyrrolidone solution were added to the mortar, respectively. After fully ground, the grinding solution was centrifuged at 4 °C, 12,000 r·min−1 for 30 min, and the middle clear liquid was filtered by using a 0.25 μm filter membrane. At room temperature (25 °C), the substrate solution (0.9 mL), borate buffer  and crude enzyme solution (0.1 mL) were added to the 1 cm quartz cuvette. Enzyme activity was determined spectrophotometrically by measuring the increase in absorbance at 234 nm over a 1 min period.Lipoxygenase (LOX) activity was determined by reference to the method of Ke et al.  with somSuperoxide dismutase (SOD), phenylalanine deaminase (PAL), ascorbate peroxidase (APX), pyruvate dehydrogenase (PDH), and succinate dehydrogenase (SDH) activity were measured according to the instructions of the Solarbio  kit.−1 at 4 °C for 30 min. Determination of PPO activity: supernatant (0.6 mL) and buffer (0.3 mL) were added to the test tube, and balanced in a water bath at 30 °C for 5 min, then 0.05% catechol (3 mL) was added, and the OD420 value was measured immediately after shaking. Record once every 30 min for a total of 5 min, an increment of 0.01 in OD420 per minute was regarded as one unit of enzyme activity. Determination of POD activity: supernatant (0.5 mL) and 0.1% guaiacol (2 mL) were added to the test tube, and test tube was equilibrated at 30 °C water bath for 5 min, then 0. 18% H2O2 (1 mL) was added, and the OD470 value was measured immediately after shaking. We recorded once every 30 min for a total of 5 min, and an increment of 0.01 in OD470 per minute was regarded as one unit of enzyme activity.Polyphenol oxidase (PPO) and peroxidase (POD) activities were measured by Cao . Enzyme p < 0.05.Statistical analyses were performed with SPSS Statistics 23 . Significant differences were ascertained using Duncan’s multiple range tests at the level of p < 0.05). On the 35th day of storage, the weight loss rate of the control group and 1.0 μL·L−1, 1.5 μL·L−1, and 2.0 μL·L−1 treatment groups were 4.58%, 1.79%, 1.81%, and 1.89%, respectively. Throughout storage, the control root mustard showed an overall decreasing trend in hardness due to nutrient depletion  (p < 0.05) (p < 0.05). In summary, this indicated that 1-MCP fumigation could alleviate the cell membrane damage of root mustard and prolong the storage time of root mustard.The cell permeability of root mustard first increases, followed by a decrease, reaching a peak at 28 days. The lowest cell permeability of root mustard was observed in the 1.0 μL·L < 0.05) A. As sho−1 gradually emerged (p < 0.05).Vit C is a hexolactone compound synthesized in plants. It is not only an essential substance for maintaining human health, but also has special functions for plants in maintaining the redox balance of cell photosynthesis and metabolism . The Vit emerged A. The li emerged B. In con−1 1-MCP having the highest moisture content  . The trends in PAL activity were similar for control and treated root mustard  A–C. On d mustard D. Howeve−1 superior to the other concentrations . The results demonstrated that 1-MCP treatment could better maintain the reduction of APX content in root mustard at the late storage stage.The activity changes of SOD, SDH, and POD showed similar trends, which increased first and then decreased. Compared to the control, 1-MCP promoted SOD and SDH activities during storage, with 1.0 μL·Ltrations A,B. The 15 U·g−1 C. At the15 U·g−1 D. In con−1 1-MCP-treated groups was 46.0% and the contribution of the second principal component was 31.1%, with a cumulative contribution of 77.1%, indicating that these two principal components basically reflected all the characteristics between the control and 1.0 μL·L−1 1-MCP-treated root mustard sample groups. The root mustard samples of CK14d and 1.0 μL·L−1 1-MCP28d were clustered together, indicating a high similarity between the first and second principal components of these two samples, which could laterally respond that the storage effect of 1.0 μL·L−1 1-MCP-treated root mustard at day 28 of storage was similar to that of the control at 14 days of storage, indicating that 1.0 μL·L−1 1-MCP could effectively prolong the storage quality of root mustard.As can be seen from −11-MCP treatment group is shown in −1 1-MCP-treated root mustard samples.The analysis of OPLS-DA in the control and 1.0 μL·LGarcinia mangostana L.) fruit [−1 had the best storage effectiveness. A study on apples has been conducted showing that 1-MCP treatment inhibited the hydrolysis activity of the cell wall, maintained the pectin of the apple cell wall, and decreased the dissolution of polysaccharides and neutral sugar to delay the softening process [Root mustard is a root vegetable with a high respiratory metabolism, which accelerates shriveling, yellowing of the rind, and loss of nutritional quality after harvest. In the studies indicated that 1-MCP has been extensively used in postharvest storage with its functions of inhibiting ethylene and extending storage life . Current.) fruit , banana .) fruit , etc. Ma.) fruit  found th process . Zkaya e process  also fou process  found th process ,53,54.−1 1-MCP could inhibit the respiration and ethylene release rate of root mustard, indicating that 1-MCP prevented the formation of the ethylene receptor complex and blocked the ethylene-induced signaling pathway.Respiratory metabolism and ethylene release are important physiological activities that provide nutrients and energy to sustain the normal vital processes in post-harvest fruits and vegetable harvests. They reflect the maturation and aging of fruits and vegetables during storage . The rolPyrus communis L. showed that 1-MCP inhibited PPO activity, delayed fruit browning, and increased SOD and POD activities [−1 1-MCP had better quality, lower PPO activity, and better POD and SOD activity.In recent years, brassica vegetables have attracted much attention because of their functions as antioxidants, preventing cancer and cardiovascular diseases and delaying the aging process . Liang etivities ,63. With−1 and 1.5 μL·L−11-MCP had better effects on promoting SDH activity and inhibiting PDH activity than 2.0 μL·L−11-MCP treatment. The results showed that as an important enzyme involved in energy metabolism, the increase in SDH activity and the decrease in PDH activity might help alleviate the senescence of mustard through 1-MCP treatment.The changes in relative conductivity and MDA content reflected the damage degree to cell membrane. When plants are injured by stress, cell membrane lipids and membrane proteins are vulnerable to damage, resulting in a large number of intracellular substances extravasation, thereby increasing the relative conductivity . MDA con−1,1.5 μL·L−1, and 2.0 μL·L−1 1-MCP were used to treat root mustard, and the changes in physiological indicators and associated enzyme activities were analyzed over the storage period. The results indicated that, compared to the control, the three groups of 1-MCP could effectively slow down the decrease in weight loss rate, hardness, lignin, and Vit C content, inhibit respiration intensity, reduce ethylene release, decrease cell permeability and MDA accumulation, maintain cell membrane integrity, inhibit the increase in LOX, PDH, and PPO activities, and maintain SDH, SOD, APX, PAL, and POD activities at a high level. The effect of 1.0 μL·L−11-MCP was better than 1.5 μL·L−1 and 2.0μL·L−11-MCP. In summary, 1.0 μL·L−11-MCP treatment delayed the senescence and quality deterioration of mustard and maintained good edible quality and nutritional value after 35 days of storage. At the same time, 1-MCP is a non-toxic, trace amount of a safe chemical preservative that is easy to handle and economical to use.In this experiment, 1.0 μL·L


Registered trials and real-world evidence (RWE) studies provided evidence on the efficacy of once-weekly (OW) semaglutide on hyperglycaemia and cardiovascular risk factors as add-on or de-novo treatment in type 2 diabetes (T2D).In a retrospective analysis of electronic data files from 258 T2D patients, this RWE study aimed to explore the impact of OW semaglutide on biochemical and anthropometric outcomes after 6 and 12 months in patients receiving at least one prescription of OW semaglutide between September 2019 and May 2021.p<0.0001 for both). At these time-points, HbA1c values were <7% in 61% and 57% of cases. HbA1c reduction was greater in patients with higher baseline HbA1c levels and it occurred irrespective of gender, age, insulin therapy and complications. The residual number of cases with HbA1c ≥9% by the study end was low (5.3% vs 18.9% at baseline). Weight loss occurred in 73.5% and 78.1% of cases and, compared to baseline, it was ≥5% in 21.2- 25.4% and ≥10% in 6.8-18.2% after 6 and 12 months, respectively. Significant predictors of HbA1c reduction after 6 months of OW semaglutide treatment were baseline HbA1c (p<0.0001), bodyweight reduction (p<0.0001) and disease duration (p<0.001), while baseline HbA1c was the only predictor of HbA1c response after 12 months (p<0.0001). Reported adverse events were consistent with the known safety profile of semaglutide.During the study period, 154 and 56 consecutive patients completed the 6 and 12 months of OW semaglutide treatment. HbA1c levels decreased by -1.02±0.1% after 6 months and -1.1±0.1% after 12 months of OW semaglutide (Real-world evaluation of weekly subcutaneous treatment with semaglutide in a cohort of Italian diabetic patients. Incretins are peptides secreted in response to meal ingestion which, at physiological concentrations, stimulate insulin release and reduce glucagon secretion. The incretin family comprises several peptide hormones, including secretin, VIP, pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon as well as glucagon-like peptides, i.e. GLP-1 and GLP-2, glycentin, oxyntomodulin and gastric inhibitory peptide (GIP) . In addiSemaglutide is a long-acting GLP-1 analog with a once-weekly (OW) extended release, developed to treat T2D patients who are unable to achieve their hemoglobin A1c (HbA1c) goals with other anti-hyperglycemic medications. Marketing of OW semaglutide has been granted in Europe since February 2018 and has been available for therapeutic use in Italy since September 2019 , 16. The2; BMI ≥ 35 kg/m2. Patients were not excluded if on other GLP-1 RA or dipeptidyl peptidase 4 inhibitors (DPP4i), which were then replaced by OW semaglutide upon enrollment. According to regulatory restrictions at the time of the study, gliflozins and short-acting insulins were withdrawn at study entry. Exclusion criteria included gestational diabetes mellitus and T1DM, previous or current diagnosis/family history of medullary thyroid carcinoma or multiple endocrine neoplasia, incomplete anthropometric and/or biochemical data at baseline and during follow-up visits. Patients were excluded from the study if showing poor compliance to OW semaglutide  or if they were suffering from ESRD requiring dialysis or kidney disease unrelated to diabetes or hypertension.This is a single-center, retrospective, observational cohort study conducted at the Endocrinology and Diabetology outpatient clinic, Ospedale Maggiore della Carità, Novara, Italy. The study included patients with an established diagnosis of T2D, who underwent OW semaglutide treatment between September 2019 and May 2021 and were followed over 12 months. The study was notified to the Ethics Committee and performed in accordance with the current legislation on Observational Studies and the Declaration of Helsinki. Patients provided their written consent to process personal data, and the publication of the study results. For this study, patients previously diagnosed with T2D aged ≥ 18 years who were on a standard-care treatment regimen with oral antidiabetic drug (OAD) or insulin therapy were considered eligible for OW semaglutide if not on target with standard OAD, i.e. HbA1c ≥ 7% (≥ 53 mmol/mol), or if any of the following inclusion criteria was accomplished: HbA1c < 7% but intolerance towards ≥ 1 OAD other than GLP-1RA; previous CVE or high/very high CVR according to ESC 2019 guidelines ; estimat2). The estimated glomerular filtrate rate (eGFR) was calculated with the MDRD [2 [Before enrollment, all patients were on stable OAD or insulin treatment for at least 3 months. At baseline, data collected for analysis included clinical history, antidiabetic drug history, anthropometric and biochemical parameters. Study measures included BMI, glucose, HbA1c, lipids, liver function tests, serum creatinine and microalbuminuria. Height was measured by the Harpenden stadiometer to the nearest mm with the subject head in Frankfurt plane and weight using electronic scale both taken in triplicate. Averaged BMI was calculated as body weight divided by squared height  and continuous variables . Further, stratification by kidney function was performed according to eGFR CDK-EPI categories .Plasma glucose levels  were measured by the gluco-oxidase colorimetric method . Routine laboratory data included total cholesterol, high-density and low-density lipoprotein cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, measured by enzymatic methods . HbA1c levels were measured by high-performance liquid chromatography (HPLC), using a Variant machine ; intra- and inter-assay coefficients of variation are, respectively, lower than 0.6 and 1.6%. Linearity is excellent from 3.2 (11 mmol/mol) to 18.3% (177 mmol/mol). Serum creatinine levels were assessed with the enzymatic method of creatinine deamidase/GLDH (Advia Chemistry-Bayer).Safety data were collected during the study period at each time visit.t-test or repeated measures ANOVA. Multivariate analysis of variance (MANOVA) was used to test the statistical significance of the effect of one or more independent variables including HbA1c variation, sex, age and duration of disease within the eGFR CDK-EPI categories (dependent variable). Correlations analyses were calculated with Pearson’s coefficient. Analysis of repeated measures was used to determine differences in subjects before and after treatment with OW semaglutide. As covariates, we assessed the effect of gender, age, BMI, disease duration, presence of diabetic complication/s, use of insulin, previous CV events, number of drugs for hypertension, use of ACE-inhibitors, angiotensin receptor blockers (ARBs), or diuretics (yes/no). A stepwise regression was used to determine the association of delta variations between HbA1c and study covariates, so as to define independent predictors of HbA1c response to OW semaglutide. Statistical significance was assumed at p < 0.05. The statistical analysis was performed with SPSS for Windows V.21.0 .Data obtained from computerized medical record system in use at Italian diabetes centers  were used for the analysis. Data are expressed as mean ± standard error of the mean (SEM), and as absolute or percent delta variations compared to baseline. Distributions of continuous variables were examined for skewness and were logarithmically transformed as appropriate. Differences between values obtained at baseline and during the follow-up were calculated by two-tailed unpaired The original cohort of T2D patients receiving at least one prescription of OW semaglutide comprised 271 people, in 258 of whom a complete dataset allowed baseline analysis , ≥ 60–89 ml/min in 115 (44.4%) and ≥ 90 ml/min in 80 patients (30.9%).At baseline, there were no differences in the tested variables between genders but for BMI, this being unexpectedly higher in females than males, as well as microalbuminuria, creatinine, lipids, ALT and urate levels Table .Of the 258 consecutive patients, 154 completed the 6-month and 56 completed the 12-month follow-up. The samples scrutinized at the different timepoints hence reflected the patients consecutively enrolled until the study end with no dropouts. Following the standard dose-escalation protocol , OW semap < 0.0001 for both) both at 6 and 12 months (Table Improvements in glycemic control were significant in terms of HbA1c and glucose levels reduction (OW semaglutide treatment determined weight loss in 73.5% of patients after 6 months (ranging between − 0.5 and − 19 kg) and 78.1% after 12 months (ranging between − 0.2 and − 26 kg). The overall reduction in bodyweight was significant both in terms of body mass and BMI values    and after 12 months  , triglycerides , and GGT levels . Associations were only significant after 6 months between delta HbA1c and disease duration  as well as ALT variation .A wide number of associations were tested to assess correlates of HbA1c response to OW semaglutide. It should be noted that signs of association reflected deltas or absolute variables. Delta HbA1c response to OW semaglutide was well correlated with baseline HbA1c levels both after 6 and 12 months of the study (th) Fig. A. Converβ =  − 0.59, p < 0.0001), delta BMI variation  and disease duration . The adjusted R2 for the model was 0.52.At stepwise multivariable regression analysis Table , the mosβ =  − 0.72, p < 0.0001)  Table , with anA total of 39 out of 258 patients (15.1%) receiving at least one dose of OW semaglutide withdrew from treatment. OW semaglutide was discontinued in 29 cases (11.2%) for gastrointestinal intolerance occurring within few weeks since therapy commencement , while treatment was discontinued due to therapeutic inefficacy in 9 patients and pregnancy in 1 case. In 3 patients (0.1%), OW semaglutide was withdrawn for poor compliance. In 9 more patients, dose increment from 0.5 mg to 1.0 mg weekly was constrained due to gastrointestinal intolerance occurring during dose titration. However, gastrointestinal side effects did not appear to contribute to the reductions in HbA1c and/or body weight.RWE studies on GLP-1RAs have provided extensive evidence that OW semaglutide reduces HbA1c and body weight irrespective of previous GLP-1 RA use, with potentially higher persistence than other GLP-1 RAs –43. Our GLP‐1 analogs are recommended as add‐on therapy to metformin and other OADs and as first injectable therapy in preference to insulin , 45. AmoThe patient population herein scheduled to receive OW semaglutide was moderately aged, averagely complicated, and predominantly obese. Our results show that OW semaglutide was associated with clinically and statistically significant reduction in mean HbA1c, regardless of age, gender, insulin therapy, prior history of GLP-1RA use and diabetic complications. HbA1c decreased on average by 1.0–1.1%, and the magnitude of response was greater in patients with higher baseline HbA1c levels. Target HbA1c values < 7% were achieved in 61% and 57% of cases after 6 and 12 months, respectively. Our results seem comparable to those of other RWE studies. While the SUSTAIN studies reported a mean HbA1c reduction from baseline of 1.0–1.5% and HbA1c < 7% in 68.7–82.8% of cases after 6–12 months of treatment , recent In addition to its anti-hyperglycemic effects, OW semaglutide outstands for its ability to reduce body weight thus aiding the global metabolic control. As a proof of this, we observed significant changes in body weight, with an average reduction of 3.1 kg after 6 months and 4.3 kg after 12 months as compared to baseline. Collectively, weight loss was ≥ 5% in 21 and 25.4% after 6 months and was ≥ 10% in 6.8 and 18.2% of cases after 12 months of treatment. Recent RWE have shown similar effects on weight . It is wBased on previous data from our group , a goal In insulin users, initiation of OW semaglutide led to a reduction in HbA1c and body as significant and comparable as in non-insulin users. Like another RWE analysis , we recoSemaglutide was generally well-tolerated, though a lower proportion of persons experiencing side effects (18%) compared to the clinical studies were noted and this was predominated by gastrointestinal side effects, such as nausea, diarrhea or constipation, and abdominal cramps. No patient was admitted to hospital due to pancreatitis or for any other reason. The figure reported here is higher than that seen in the SUSTAIN-6 trial (13.1%) and some RWE studies , 32, whiThis study presents limitations that are typical of retrospective observational studies, such as the lack of comparators and randomization, the access to utilization, and cost of health resources. Moreover, the low number of patients at the 12-month follow-up may hamper the conclusions of our analysis. It should also be acknowledged that the 1.0 mg dose was prescribed to only 22% of cases, implying that up-titration was incomplete in most cases possibly due to the choice of clinicians, concomitant therapies of the population sample, and underlying intentions to limit occurrence of side effects. Nevertheless, the effect of treatment on HbA1c and weight was similar to that reported in most RWE studies –31. Poin


Lactiplantibacillus plantarum could be modulated through the modification of fermentation parameters. Due to their high nutritional value, agri-food by-products could be considered a useful fermentation source for microorganisms. Therefore, these by-products were proposed as fermentation substrates to produce GABA in this study. Previously, several experiments in Man Rogosa Sharpe (MRS) broth were performed to identify the most critical parameters to produce GABA using the strain Lactiplantibacillus plantarum K16. The percentage of inoculum, the initial pH, and the concentration of nutrients, such as monosodium glutamate or glucose, significantly affected the biosynthetic pathway of GABA. The highest GABA yield was obtained with 500 mM of monosodium glutamate and 25 g/L of glucose, and an initial pH of 5.5 and 1.2% inoculum. Furthermore, these investigated parameters were used to evaluate the possibility of using tomato, green pepper, apple, or orange by-products to get GABA-enriched fermented media, which is an excellent way to revalorise them.Probiotic metabolites, known as postbiotics, have received attention due to their wide variety of promoting health effects. One of the most exciting postbiotic is gamma-aminobutyric acid (GABA), widely produced by lactic acid bacteria, due to its benefits in health. In addition, the performance of the biosynthesis of GABA by  These health benefits can be produced because of the positive interaction between probiotics and the host gut microbiota, triggering the activation of different intracellular signalling pathways3. For example, the activation of genes involved in the synthesis of mucin avoids pathogens' adhesion to the gut barrier, the enhancement of phagocytosis through the increase of macrophages or the attenuation of pro-inflammatory cytokines production4.Probiotic microorganisms are now widely consumed worldwide due to their potential to preserve and enhance human health9. A wide range of compounds could be classified as postbiotics, such as vitamins, minerals, amino acids, neurotransmitters, or lipid compounds10. One of the most promising postbiotic is the neurotransmitter gamma-aminobutyric acid (GABA)12. This compound can reduce anxiety and depression in humans, influence several neurochemical pathways, enhance the immune system, or modulate blood pressure decreasing the likelihood of developing heart problems13.Likewise, probiotics can also produce host benefits by metabolising different nutrients and producing bioactive compounds classified as postbiotics which can be defined as metabolites synthesised by these microorganisms or other compounds released during fermentation processes14. However, the poor synthesis performance, the detrimental effect on the environment, and the low profitability of the process led to the substitution of the chemical production with a more suitable production by using a biotechnological process carried out by microorganisms15. Some examples of interesting GABA producers are lactic acid bacteria (LAB)16, Bacillus subtilis17, Aspergillus oryzae18, Listeria monocytogenes19or Bifidobacterium20. Among these microorganisms, LABs have been considered one of the most attractive alternatives to synthesise GABA due to the high performance of their biosynthetic process, their classification as generally regarded as safe (GRAS) microorganisms and their potential beneficial effects on human health21.Consistent with the health benefits of GABA, this compound was initially produced industrially by chemical synthesis to meet pharmaceutical and food companies’ demandsl-glutamic acid (l-Glu) is decarboxylase by a GAD enzyme resulting in the production of a GABA molecule22. Usually, the GAD enzyme is encoded by a gadB gene, but some LAB can present two genes such as Levilactobacillus brevis which has a gadA and gadB gene. Moreover, some species, such as Lactobacillus buchneri, Lb. curvatus or Lb. sakei, could even present potential transcriptional regulators that can enhance the synthesis of GABA21. For instance, Gong et al.23 highlighted how the transcriptional regulator GadR presented in L. brevis is directly linked to the high GABA yield and the resistance against acid environments of this bacteria. Due to the diversity of genetics involved in the GAD system, the GABA yield could be very different between species such as, L. buchneri WPZ001 yielded 117 g/L of GABA24, L. brevis NCL912 produced 103.7 g/L of GABA25 or Lactiplantibacillus plantarum (Lactobacillus plantarum) N5 yielded 21.8 g/L of GABA26. Cui et al.21 explained that the GAD systems is strain-specific and even strains with the same GAD system could have different yield of GABA.The synthesis of GABA is commonly performed through the glutamic acid decarboxylase pathway (GAD) as a mechanism triggered under stressful environments. Specifically, a molecule of L. plantarum strains could produce a great amount of GABA, depending on the source where they were isolated from, and the yield of the machinery involved in the biosynthetic pathway of GABA27.Within LAB, 28. Several studies have been conducted to adjust the main physic-chemical parameters involved in the GABA synthesis. For instance, Sharafi and Nateghi29 optimised the GABA production by L. brevis by studying the effect of temperature, initial pH, l-Glu, concentration and fermentation time. They obtained that the fermentation carried out at 34 °C, with an initial pH of 4.65, 650 mmol of l-Glu and for 96 h of incubation time, enhanced more than twice the synthesis of GABA compared to non-optimised conditions. Wu et al.30 and Song and Yu31 reported that the inoculum percentage and the nitrogen and carbon source type could positively influence the GABA synthesis by Lactobacillus strains.GAD pathway performance can be modulated by adjusting several environmental parameters such as temperature, initial pH, or oxygen availability. In addition, the type and concentration of minerals, coenzymes, nitrogen, carbon sources, and other additives could positively influence GABA biosynthesisLactobacillus growth. However, the wide variety of nutrients used in this culture media increases the cost of the production process. Thus, it is not considered a suitable fermentation media for scale-up production32. During the last years, by-products from the agri-food industry has gained attention to be used as low-cost fermentation media which puts a value on potential pollutants.Furthermore, these optimisation processes are generally performed using MRS broth, characterised by the high concentration of nutrients necessary for 33 reported that from 900 million metric tons of fruit production in 2020, approximately a 30% was discarded, normally producing a strong environmental impact because these by-products are normally burned or placed in landfills34, although it is also being used to produce animal feed35. Consequently, the use of these agri-food by-products as culture media for fermentation processes can be a good way to revalorise this type of waste, as well as to produce bioactive compounds useful for the formulation of new drugs and functional foods36.In general, agri-food industries generate a considerable amount of waste mainly produced from the transformation of raw fruits and vegetables into final products like juices or smoothies, which normally discard structural parts such as seeds, peels, leaves, or pulps. Mnisi et al.37 proposed to use molasses, dairy sludge, and soybean meal as fermentation media to produce GABA by L. brevis, Limosilactobacillus fermentum and L. plantarum. Zarei et al.38 made a functional drink using whey protein, considered a high environmental impact waste product, as the primary source to synthesise GABA by L. plantarum. In our previous study, L. plantarum K16 was isolated from Kimchi and identified as GABA-producer. Then, it was evaluated how parameters such as temperature, the concentration of yeast extract and incubation time influenced the GABA production by L. plantarum K1639 Therefore, the objective of this study was to continue with the analysis of parameters, such as inoculum percentage, initial pH, monosodium glutamate (MSG) concentration, and glucose concentrations, involved in the GABA production of L. plantarum K16 using MRS broth and achieve the highest yield of GABA in this medium. Afterwards, a fermentation trial was performed to determine if tomato, green pepper, apple, or orange by-products could be considered as suitable fermentation substrates to obtain GABA-rich fermented products.Falah et al.l-Glu, and the supernatants were collected to analyse the GABA content using ultra-high-performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS). Only one of the isolated LABs was able to produce GABA which was identified as L. plantarum K16. Therefore, L. plantarum K16 was used to evaluate how different parameters could modulate the synthesis of GABA.LABs were isolated from kimchi through standard culturing methods in the Food Biotechnology laboratory . The isolated LABs were grown in MRS broth supplied with L. plantarum K16 strain was carried out in several stages following a one-factor-at-a-time (OFAT) experimental design in MRS broth . Therefore, the optimisation process was performed by studying different levels of one fermentation parameter while keeping unchanged the other fermentation parameters. The beginning of this optimisation process was explained in a previous study39, where the incubation temperature, concentration of yeast extract and fermentation time were evaluated. The results of this experiments indicated that the initial conditions to continue the optimisation process should be MRS broth supplied with 5 g/L of glucose, 12 g/L of yeast extract, an initial pH of 5.5, inoculum of 1%, 500 mM of MSG, incubation temperature of 34 °C and 96 h of fermentation. Furthermore, in the current research the fermentation parameters studied in MRS broth were inoculum percentage, initial pH, MSG concentration and glucose concentrations. For each experiment, an inoculum of L. plantarum K16 was prepared in MRS broth overnight at 37 °C. Then, the amount of GABA production  was quantified by UHPLC-MS. Likewise, the microbial growth was measured by plating serial dilutions in MRS agar and counting colonies to calculate the colony-forming units (CFU) and expressed as log CFU/mL (± 0.01). Finally, the pH value of the fermented medium was measured (± 0.1) with a Crison Basic 20 pHmeter .The optimisation process for GABA synthesis by 40, different percentages of L. plantarum K16 strain were used in the fermentation process. The fermentation media was prepared, with 250 mL Erlenmeyer flask containing 100 mL of working volume, by adding 5 g/L of glucose to MRS broth composed by 12 g/L of yeast extract, adjusted to an initial pH of 5.5 and sterilised by autoclaving the culture medium at 121 °C for 15 min. Afterwards, the medium was enriched with 500 mM of sterilised MSG, further inoculated with 0.8, 1.0, 1.2 and 1.4% of L. plantarum K16 strain and incubated at 34 °C without shaking. After 96 h of fermentation, analytical samples of the fermented medium were taken to determine the pH, GABA amount and the CFU/mL.According to the research of Kantachote et al.The MRS broth was prepared as previously described for glucose, yeast extract and MSG concentrations, and 4.0, 4.5, 5.5 and 6.0 as different initial pH values. An inoculum percentage of 1.2% was selected as the optimum value obtained for GABA production from the previous stage, and the fermentation medium was incubated at 34 °C during 96 h. Likewise, analytical samples of the fermented medium were taken to determine the pH, GABA amount and the CFU/mL.L. plantarum K16 strain, and the medium was incubated at 34 °C during 96 h. Also, analytical samples of the fermented medium were taken to determine the pH, GABA amount and the CFU/mL.Different MSG concentrations  were evaluated to determine how this precursor of the GAD biosynthetic pathway could influence the production of GABA. The fermentation media was prepared by adding 5 g/L of glucose to MRS broth composed by 12 g/L of yeast extract. Following the results obtained in the previous OFAT stages, the initial pH was fixed to 5.5, the medium was inoculated with 1.2% of 41. Different glucose concentrations  were tested in the MRS broth containing 12 g/L of yeast extract and 500 mM of MSG. This MSG concentration was selected as the optimum value obtained for GABA production from the previous section. As other assays, initial pH was adjusted to 5.5, the medium was inoculated with 1.2% of L. plantarum K16 strain and incubated at 34 °C for 96 h. Moreover, analytical samples of the fermented medium were taken to determine the pH, GABA amount and the CFU/mL.According to the scientific literature, glucose was chosen as the best carbon source for the optimisation of GABA production by LAB fermentationL. plantarum K16. Thereafter, different agri-food by-products such as tomato, green pepper, apple, and orange pulp and seeds (obtained from private suppliers) were selected to be used as fermentation substrates to produce GABA . Table 42. The fermentation media from agri-food by-products were firstly prepared by grinding and re-suspending independently 5 g of each by-product into distilled water by stirring. Due to the importance of glucose and yeast extract in L. plantarum K16 to produce GABA, the media was enriched with extra 25 g/L of glucose and 12 g/L of yeast extract. Subsequently, the pH was adjusted to 5.5 and the medium sterilised by autoclaving43. After the sterilisation, these agri-food by-product media were supplied with 500 mM of the precursor MSG, inoculated with 1.2% of L. plantarum K16 strain and incubated at 34 °C during 96 h. As before, analytical samples of the fermented medium were taken to determine the pH, GABA amount and the CFU/mL.The previous studies carried out in MRS broth helped to evaluate how different fermentation parameters could influence and improve the production of GABA by An ACQUITY UPLC H-class system  with a HILIC column  (Waters) coupled with a SecurityGuard ULTRA Cartridge pre-column (Waters) was used for the analysis of GABA in the different fermented medium samples. Column temperature was set to 30 °C, sample temperature was set to 10 °C, and injection volume was 3 µL. An isocratic elution with a mixed in volume of 5% of acetonitrile  and 95% of 0.1% formic acid  prepared in Milli-Q water as mobile phase, and a flow rate of 0.25 mL/min, was used. A triple quadrupole MS equipped with an orthogonal electrospray ionisation (ESI) source  was used for detection. The instrument was operated in positive mode electrospray (ESI +), MS settings were used as follows: capillary voltage 3.05 kV, desolvation temperature 400 °C, source temperature 120 °C, cone and desolvation gas (nitrogen) flow 60 L/h and 800 L/h, respectively, and collision gas (argon) flow 0.10 mL/min. High purity nitrogen and argon were used . MS was run in multiple reaction monitoring (MRM) including two ion transitions for GABA: m/z 104 > 87 for quantification and m/z 104 > 69 for identification. Data acquisition and quantification were performed using MassLynx software version 4.1 (Waters). Quantification was performed against a linear (1/x weighted) regression curve based on duplicate injections of calibration GABA standard solutions.L. plantarum K16 strain among the fermented media within each fermentation parameter studied. Bonferroni's method was applied for pairwise comparison, and statistical significance was declared at P ≤ 0.05. In addition, Rho Spearman correlation coefficient was calculated to investigate the relationship between the amount of GABA produced and the nutritional composition of each agri-food by-product used.The statistical analysis was carried out using the IBM-SPSS statistics software version 25.0 . One-way analysis of variance (ANOVA) was used to evaluate the presence of statistically significant differences in the amount of GABA produced and the growth of Different initial inoculum percentage, 0.8% (7.41 ± 0.07 log CFU/mL), 1.0% (7.44 ± 0.06 log CFU/mL), 1.2% (7.50 ± 0.03 log CFU/mL) and 1.4% (7.60 ± 0.08 log CFU/mL), were assessed to determine the suitable concentration for producing the greatest GABA. The results, represented in Table P ≤ 0.05) up to 951.05 ± 49.26 mg/L, together with a slight decrease in the media pH up to 4.0. Furthermore, a significant increase (P ≤ 0.05) in the amount of GABA was obtained when the initial pH was 5.5 reaching the maximum value of GABA produced (1419.93 ± 57.47 mg/L). Contrarily, when the initial pH raised to 6.0, a substantial decrease (P ≤ 0.05) in the amount of GABA (1323.01 ± 72.08 mg/L) was observed compared with the value observed when the initial pH was 5.5. At the same time, the increase of GABA concentration during 96 h of fermentation was accompanied by a decrease in the growth of L. plantarum K16 strain, hitting the concentration of 7.31 ± 0.14 log CFU/mL when the initial pH was 5.5.Several initial pH, between 4.0 and 6.0, was studied, focusing on identifying the most suitable to enhance the GABA synthesis. In this case, after 96 h of fermentation, a concentration of 197.5 ± 11.92 mg/L of GABA and no changes in the pH medium were observed using an initial pH of 4.0 (Table P ≤ 0.05) in the GABA yield by L. plantarum K16 strain (Table P ≤ 0.05) up to 1207.14 ± 60.38 mg/L when the concentration of MSG was 300 mM. The maximum GABA production (1419.93 ± 57.47 mg/L) was reached at 500 mM of MSG concentration, although a significant decrease (P ≤ 0.05) in the amount of GABA was observed at MSG concentration greater than 500 mM (1027.81 ± 38.21 mg/L). On the other hand, no significant variation (P > 0.05) in the microbial growth was observed when the MSG concentration was higher than 300 mM  of GABA synthesis (1391 ± 64.84 mg/L) was observed when the glucose concentration reached 23 g/L in the medium. The maximum concentration of GABA (2115.70 ± 73.83 mg/L) was observed with 25 g/L of glucose, but a higher concentration of glucose (27 g/L) resulted in a significant decrease (P ≤ 0.05) in the amount of GABA produced (1771.6 ± 63.61 mg/L) (Table P > 0.05) maintaining viable counts around 7 log CFU/mL  in the microbial growth reaching a concentration of 8.88 ± 0.14 log CFU/mL. Green pepper and tomato by-products significantly (P ≤ 0.05) enhanced the biosynthetic pathway of GABA producing 1626.52 ± 55.9 mg/L and 1776.75 ± 109.49 mg/L, respectively (Table L. plantarum K16 was significantly higher (2115.7 mg/L) compared to the values observed using agri-food by-products.A production trial of GABA was performed using different kinds of agri-food by-products as fermentation substrates for L. plantarum K16 to produce GABA. When the percentage of inoculum was assayed, a significant increase in the amount of GABA was observed using an inoculum of 7.5 log CFU/mL (1.2%) compared to using an inoculum of 7.41 log CFU/mL (0.8%) (Table 40 showed that L. plantarum DW12 produced the highest concentration of GABA (128 mg/L) when the initial inoculum was 7 log CFU/mL giving a microbial cell growth of 8.01 log CFU/mL. However, a higher inoculum (8 log CFU/mL) increased the microbial cell growth to 9.2 log CFU/mL but yielded 101 mg/L of GABA. Rayavarapu et al.28 showed that the highest amount of GABA produced by L. fermentum was 3.79 g/L and the microbial cell growth was 5.8 log CFU/mL using a 1% of inoculum. However, an increase of inoculum to 2% did not significantly change the GABA production yielding 3.71 g/L and a microbial cell growth of 6.4 log CUF/mL. Even lower GABA synthesis was observed when the inoculum used was 3 or 4% obtaining 2.62 and 2.12 g/L of GABA, respectively.The first aim of this research has focused on identifying fermentation parameters involved in GABA synthesis. Therefore, an OFAT experiment was carried out in MRS broth to evaluate how the percentage of inoculum, initial pH, MSG, and glucose concentration influence CFU/mL 1.% compare44. Consequently, LAB have developed protective mechanisms to avoid cell damage45. In this sense, Heunis et al.46 identified about 300 proteins involved in the protection of L. plantarum 423 strain against acid stress. Most of protective mechanisms try to maintain the intracellular pH using proton pumps, decarboxylation, deamination, metabolism changes, or strengthening the cell envelop. Fernández and Zúñiga47 highlighted the importance of the catabolism of amino acids, such as aspartic acid, arginine or glutamic acid, as critical coping mechanism to overcome stressful environments. The GAD pathway is considered one of the most essential acid tolerance systems, which is based on the decarboxylation of glutamic acid by a GAD enzyme resulting in a molecule of GABA, classified as an alkaline compound48. In addition, during GAD pathway, a cytoplasmic proton is consumed increasing the internal pH and improving cell homeostasis maintenance49. Shin et al.50 indicated that the catalytic activity of GAD enzyme is extremely dependent on pH, and the optimum pH value significantly enhance the relative activity of the enzyme and thus the GABA yield. In our study, the highest GABA production  by a L. plantarum strain isolated from fermented fish products.Regarding the initial pH of the culture medium, LAB are broadly adapted to a wide range of pH values mainly due to the acid stress caused by their metabolism, because LAB normally produce a wide amount of lactic acid from carbohydrates fermentation. A high concentration of lactic acid creates a stressful environment in the medium that could negatively influence bacterial development and cause growth inhibition, while nutrients are still available, as well as increase cell death53. Hence, the development of L. plantarum strains could be linked to the supplementation of amino acids because, in many cases, these bacteria are unable to produce these compounds. For instance, L. plantarum could need l-Glu supplementation to metabolise it and enhance the bacteria growth54. Likewise, l-Glu could also be required to activate the secondary metabolism to produce postbiotic compounds such as GABA55 or plantaricin56. Furthermore, l-Glu is usually supplemented directly into the fermentation media of L. plantarum strains due to this amino acid is the GABA precursor57. In the same way, MSG has been used in several studies to enhance GABA synthesis60. However, the MSG concentration should be optimised for each strain due to an excessive MSG concentration could be toxic and suppress the GAD enzyme55. In this investigation, increasing the concentration of MSG from 100 to 500 mM significantly enhanced GABA synthesis, but a reduction in GABA production was observed by supplying 550 mM of MSG  using a glutamate concentration of 500 mM. However, since MSG concentration is strain-dependent, other studies performed with L. plantarum strains reported that 80 and 200 mM of MSG were optimal for GABA synthesis62. Similarly, Yogeswara et al.58 studied the GABA production of L. plantarum FNCC 260 strain using a wide range of MSG concentrations. The results showed a maximum GABA production (1226 mg/L) by supplying to MRS broth with 100 mM of MSG. Gomaa63 required a concentration of 750 mM MSG to get the maximum GABA yield (14.5 g/L) using L. plantarum DSM749 strain isolated from Egyptian dairy products. Among other LAB species, the optimum amount of MSG can be also highly variable. Villegas et al.64 studied the GABA production using an L. brevis strain isolated from quinoa sourdough. MRS medium was supplied with concentrations of MSG up to 400 mM, reaching the highest concentration of GABA (26.29 g/L) with 270 mM of MSG. Likewise, Wu et al.30 increased the efficiency of the GABA synthesis by L. brevis RK03 strain reaching 62.53 mg/L of GABA by supplying 650 mM of MSG to the fermented medium.Generally, LAB are considered nutritionally fastidious microorganisms, which need the supplementation of vitamins and amino acids required for a proper metabolism performanceSG Table . Harnent65. In this regard, glucose is considered the most attractive carbohydrate commonly used to enhance bacterial cell growth and lactic acid production67. Moreover, glucose catabolism produces severe acidification of the medium that could trigger the activation of the GAD pathway and thus, the stimulation of GABA synthesis68. In the present study, when the MRS broth contained 25 g/L of glucose, L. plantarum K16 synthetised the great concentration of 2115.7 mg/L of GABA. Furthermore, Hussin et al.69 reported the highest GABA synthesis by L. plantarum Taj-Apis362 using 20 g/L of glucose. However, L. plantarum EJ2014 only required 10 g/L of glucose to yield 19.8 g/L of GABA70 and L. plantarum KCTC3103 showed the maximum GABA production (670 mg/L) using 5 g/L of glucose71. Contrary, Zareian et al.72 using L. plantarum MNZ strain isolated from fermented soybean showed the highest GABA (408.36 mg/L) biosynthesis when 60 g/L of glucose were supplied to the fermentation media.The source of sugar is also essential for LAB species to produce energy and cell biomassL. plantarum K16. The maximum concentration of GABA (2115.7 mg/L) was obtained using MRS broth composed of 25 g/L of glucose, 12 g/L of yeast extract, 500 mM of MSG, an initial pH of 5.5, an inoculum of 1.2%, incubated at 34 °C and fermented for 96 h. Therefore, after identifying the best conditions to produce the maximum amount of GABA by L. plantarum K16 in MRS broth, a fermentation trial was performed to assess the ability of this bacteria to produce GABA in agri-food by-products. According to the nutritional and functional value of orange, green pepper, tomato, and apple, their pulp and seeds by-products were considered suitable raw materials for fermentation. Several authors74 have proposed recycling apple waste by using it as a fermentation substrate due to its high concentration of magnesium, calcium, fibre, and phenolic compounds like flavonoids or hydroxycinnamic acid. Moreover, more than half of the raw material from the orange juice industry are wasted, which means the loss of a good source of dietary fibre, phenolic compounds, and minerals76. Likewise, pepper and tomato by-products are also considered good sources of dietary fibre, phenolic compounds, proteins, carbohydrates, and lipids78. Likewise, Table l-Glu (335 mg/100 g), followed by green pepper, orange, and apple by-product. Despite the nutritional variability between these four by-products, in the present study, they were enriched with 25 g/L of glucose, 12 g/L of yeast extract and 500 mM of MSG, to ensure that at least L. plantarum K16 had enough nutrients to synthetise GABA. Furthermore, L. plantarum K16 produced great amount of GABA reaching a concentration of 1166.81 mg/L, 1280.01 mg/L, 1626.52 mg/L and 1776.75 mg/L in apple, orange, green, and tomato by-products, respectively  using cassava, but it was lower concentration than the one observed in MRS broth (1.53 g/L). Contrarily, Moo-Chang et al.80 showed that L. sakei B2-16 in MRS broth enriched with 4% of sucrose, 1% of yeast extract and 5% of MSG (conditions previously optimised) could produce 28.05 g/L of GABA. However, significantly higher concentration of GABA, 68.05 g/L, was obtained using the by-product rice bran extract enriched with 4% of sucrose, 1%yeast extract and 12% of MSG.The fermentation process in MRS broth helped identify the essential parameters to produce GABA by l-Glu (≥ 0.9). This could mean that agri-food by-products with high content of sugar and carbohydrates could enhance metabolic pathways involved in cell duplication. Nevertheless, a higher protein and l-Glu concentration could enhance the GAD pathway.In the present study, the difference in GABA yield between each agri-food by-product could be related to the variability in their nutritional composition. Regarding Table L. plantarum K16 metabolism. For example, several compounds could activate other metabolic pathways on L. plantarum K16 strain by focusing more on these biochemical processes than on the GAD pathway. Several studies have reported the importance of other metabolic routes that protect LAB under stressful conditions such as arginine or agmatine deaminase pathways or aspartic acid or histidine decarboxylation processes82. Therefore, after confirmed that tomato, orange, apple, and green pepper by-products could be used to produce GABA by L. plantarum K16. Further research is necessary to characterise the composition of each by-product and design a specific optimisation process for each by-product to maximise the GABA production of L. plantarum K16.Furthermore, the different production of GABA, between MRS broth and by-products, could be due to agri-food by-products present a wide and great variety of compounds compared to MRS broth, which composition is fully controlled. Thus, the variability of compounds in each agri-food by-product could have different effects on L. plantarum K16 strain. The optimisation of the percentage of inoculum, the initial pH, MSG, and glucose concentration, strongly influenced the GAD pathway of L. plantarum K16 and significantly increased the GABA production in MRS broth. Afterwards, GABA production was successfully achieved using tomato, green pepper, apple, and orange by-products by applying previously optimised fermentation parameters.A wide range of relevant parameters involved in the GABA production were individually studied to achieve the highest yield of


The sperm reserves per testis (SR), sperm reserves per gram testis (SRG), daily sperm production per testis (DSP), and daily sperm production per gram testis (DSPG) were  million sperm cells, respectively. The cauda epididymal sperm reserves were 1,386 ± 456 million sperm cells, and the sperm motility (M%), morphologically normal viable cells (N%), dead sperm cells (D%), and morphologically abnormal sperm cells (A%) were 89 ± 3.2, 70.13 ± 8.0, 13.55 ± 5.05, and 16.33 ± 7.9, respectively. In conclusion, testicular morphometric parameters in Ghanaian West African Dwarf rams are highly and positively correlated (r = 0.7487–0.9354) with sperm reserves and production.The current study was conducted to investigate the association between testicular morphometric parameters and sperm reserves in Ghanaian West African Dwarf rams. The intact scrotum along with testes and epididymides from twelve rams were collected immediately after slaughtering and transported to the Regional Veterinary Laboratory. The left testes and epididymides from all rams were dissected and the testes were individually weighed. The individual length, width, and volume of the testis and cauda epididymis were determined. Tissue samples were taken from testicular parenchyma, homogenized, and used to determine gonadal sperm concentration using a hemocytometer. Spermatozoa were recovered from the cauda epididymis by the incision method and used to determine the spermiogram. The weight, length, width, and volume of the testis were 63 ± 11 gram, 6.7 ± 0.44 cm, and 4.6 ± 0.40 and 74 ± 19 cm Small ruminant production plays an important role in the economies of developing countries. The major breeds of sheep reared in Ghana include the West African Draft (WAD) or Djallonke, long-legged Sahelian, and crossbreeds. The sheep population is estimated to be 4,335,000 . IndigenEvaluation of sperm reserves includes the assessment of gonadal and extragonadal sperm reserves, which are crucial in the selection of potential good breeders . ExtragoSperm reserves have been evaluated from slaughtered domestic animals , while sThe study was conducted from May to June 2021, at the Akwatia Line slaughter slab, located in the centre of the Kumasi Metropolis. Kumasi is the regional capital of the Ashanti region and is located geographically within the forest zone of Ghana. It is a commercial city, where most animals brought for slaughtering, originate from Northern Ghana.2, where L represents the length and W is the width of the testis.The scrotal sacs from twelve clinically healthy WAD rams, aged 18–24 months, were collected immediately after slaughtering. The testes were collected after slaughtering because this procedure was less expensive. Moreover, the rams were humanely slaughtered at an approved slaughter facility where slaughtering processes adhered to laid down animal welfare principles. The procedures used in this study were approved by the animal ethics committee of the university. The body weight and age of the selected rams were estimated before slaughtering. The collected samples were packaged aseptically in polythene bags and transported on an ice chest to the Regional Veterinary Laboratory for analysis, within one hour after slaughtering. The left testes and epididymides were dissected, freed from all connective tissue and fats, and the testes were weighed individually with an electronic weighing scale . The length and width of the left testis and cauda epididymis in each sample were measured using Vernier calipers, while their respective volumes were estimated using the modified formula of a prolate spheroid  as folloSegments of testicular parenchyma were taken from the proximal and distal regions, weighed, and then homogenized with a high-speed blender for 2 min with 20 ml normal saline solution containing antibiotics  to prevent bacterial growth. The homogenate was stored overnight at 5°C to allow sperm cells to ooze out of the tissues. An aliquot of this homogenate was filtered and used to determine the gonadal sperm concentration, using an improved Neubauer hemocytometer after appropriate dilution in 0.05% formal saline as previously described . The speSpermatozoa were collected from the cauda epididymis by multiple longitudinal incisions into its parenchyma, followed by immersion into a Petri dish containing 2.9% sodium citrate buffer solution and placed in a water bath at 37°C for 30 minutes . Cauda er).The general linear model was used to analyse the results. The results obtained were expressed in the form of mean ± SD, using the GraphPad Prism software . The ass3, respectively, while the length, width, and volume of the cauda epididymides were 3.2 ± 0.35 cm, 1.8 ± 0.16 cm, and 5.48 ± 1.5 cm3, respectively.The data for morphometric parameters of the testes and cauda epididymides of WAD rams are presented in The mean values of sperm reserve of whole testis (SR), sperm reserve per gram testis (SRG), daily sperm production per testis (DSP), and daily sperm production per gram testis (DSPG) were 11,740 ± 386, 250 ± 6, 2350 ± 7.7, and 50 ± 1.0 million sperm cells, respectively .r = 0.8262–0.9676) with the weight, length, width, and volume of testes and also with sperm reserves and sperm production. Testicular morphometric parameters correlated highly and positively with each other (r = 0.7487–0.9354) and also with sperm reserves and sperm production. Sperm reserves correlated positively and highly (r = 0.9804–1.000) with sperm reserves per gram testis, daily sperm production, and daily sperm production per gram testis.The correlations between testicular morphometric parameters and sperm production are illustrated in The cauda epididymal sperm reserves were 1,386 ± 456 million sperm cells, while the sperm motility (M%), morphologically normal viable cells (N%), dead sperm cells (D%), and morphologically abnormal sperm cells (A%) were 89 ± 3.2, 70.13 ± 8.0, 13.55 ± 5.05, and 16.33 ± 7.9, respectively .Our results in terms of length and width of the testis were similar to the values of 6.3 ± 0.16 cm and 4.4 ± 0.11 cm, respectively, as previously documented in WAD rams in North Benin . However3, respectively, reported for WAD rams in Akwa Ibom State, Nigeria [The mean values of weight and volume of the testis of rams in the current study were higher than 51.20 gm and 53.7 cm Nigeria , 20. The Nigeria .However, the mean values of testes' weight, length of testes, and cauda epididymides were lower than 99.8 ± 2.6 gram, 8.7 ± 0.2 cm, and 4.4 ± 0.7 cm, respectively, as reported for Yankasa rams reared in Zaria, Nigeria . MoreoveThe mean values of SR, DSP, and DSPG in this study were higher than 2,150 ± 110, 600 ± 10, and 9.4 ± 2 million sperm cells, respectively, reported for WAD rams in Nigeria . These dThe testicular morphometric parameters highly correlated with each other and with sperm reserves and daily sperm production. Our findings agree with previous reports that high correlations existed between testicular weight, sperm reserves, and daily sperm production in WAD rams . MoreoveThe mean values of cauda epididymal sperm reserves in this study were higher than reported for WAD rams in Ibadan, Nigeria , and YanThe mean values of percent sperm motility, sperm viability, and morphological abnormal sperms in this study were within the ranges of 66.25–85.66, 84.17–87.66, and 11.84–12.96, respectively, as documented for WAD rams in Ibadan, Nigeria . On the r = 0.7487–0.9354) with sperm reserves and production. Future research should be conducted to investigate the fertilizing ability of spermatozoa recovered from recently slaughtered animals.In conclusion, testicular morphometric parameters in Ghanaian West African Dwarf rams were highly and positively correlated (