Effect of Antioxidants on Pig Semen Cryopreservation to Preserve Sperm Fertility after Thawing

Boar semen cryopreservation has a high potential in the swine industry, allowing the large-scale use of genetically superior animals, improving efficiency, product quality, helping to reduce the risk of disease spread and gathering needs from the market. From a genetic point of view, semen freezing is desirable for genetic diversification, favouring a more efficient reproduction as well as the constitution of germplasm banks, including for repopulation in case of disease outbreak. However, freezing this semen for long periods for practical use is limited by the reduced viability and fertilization potential caused to sperm during the cryopreservation process and consequently low conception rates and smaller litters after artificial insemination. In part, the decrease in the fertilizing power of frozen spermatozoa may be associated with oxidative damage due to excessive formation of Reactive Oxygen Species (ROS), osmotic stress and cell damage due to ice formation during cryopreservation. To suppress the damage caused by ROS, the present study was conducted to determine the impact of supplementation with three antioxidants, these being ascorbic acid, a-tocopherol and reduced glutathione, evaluating the parameters of semen quality, viability, total and progressive motility, vigour and agglutination rate after thawing. For this purpose, semen was collected from five boars, each being collected three times, at weekly intervals, always at the same time. Immediately after harvesting, the macroscopic (colour, appearance, and volume) and microscopic evaluation of the semen (mass motility, concentration, progressive individual motility, spermatic vigour and spermatic morphology) were evaluated. Subsequently, the semen was placed at 15°C for two hours and centrifuged at 800 x g for 10 minutes also at 15°C, removing the supernatant. For the freezing medium, a base medium consisting of a commercial MR-A extender, supplemented with 3% v/v glycerol, 10% v/v egg yolk and 0.20% w/v Sodium Dodecyl Sulfate (SDS) was used. The nine treatments used in the study were, respectively, ascorbic acid at concentrations of 100, 200 and 400μL, a-Tocopherol at concentrations of 200, 400 and 800μM and reduced Glutathione at concentrations of 2.5, 5 and 10 mg/l and numbered as T1 to T9, respectively. In the control group, semen was frozen in a medium without adding any antioxidant. The semen belonging to the different treatments and to the control was placed in 0.25ml insemination French straws and incubated at 6°C for two hours. The subsequent freezing was carried out in nitrogen vapours (-120°C) for ten minutes and immersed in liquid nitrogen after this period. After 7 days, the semen was thawed in a water bath at 37°C for 20 seconds, the straws dried on paper, placed on a microscope slide heated to 37°C and evaluated according to the parameters described above. Regarding the comparison between the different treatments, it was observed that the sperm viability obtained in the treatments with ascorbic acid as well as glutathione reduced, was not statistically different from the control group. Higher values of ascorbic acid and reduced glutathione reduced sperm viability after thawing. As for the use of a-tocopherol at a concentration of 400μM, the best results of the entire study were obtained, with sperm viability of 31.52% (±1.50). Regarding sperm motility and agglutination rate, a-tocopherol also showed the best results at the concentration of 200μM, in which the mean sperm motility was 2.57 ± 0.15 and 2.07 ± 0.15, respectively. The results of the present study allow us to infer that the addition of 200μM or 400μM of a-tocopherol to the swine semen-freezing medium has a positive effect on sperm viability parameters after thawing.

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Does supplementation of vitamin C, reduced glutathione or their association in semen extender reduce oxidative stress in bovine frozen semen?

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-11293 ◽

2020 ◽

Vol 72 (1) ◽

pp. 9-17 ◽

Cited By ~ 1

Author(s):

S.C.C. Pinto ◽

D.S. Almeida ◽

M.B.R. Alves ◽

S.A. Florez-Rodriguez ◽

G.S. Abreu Júnior ◽

...

Keyword(s):

Oxidative Stress ◽

Vitamin C ◽

Sperm Motility ◽

Reduced Glutathione ◽

Control Group ◽

Mitochondrial Potential ◽

Semen Cryopreservation ◽

Bovine Semen ◽

Frozen Semen ◽

Semen Extender

ABSTRACT The aim of this study was to evaluate the addition of vitamin C, reduced glutathione and the association thereof to the bovine semen cryopreservation extender. The ejaculate from nine bulls were divided into four fractions, each corresponding to a treatment, namely: control group-semen diluted with Tris-yolk extender; vitamin C group-semen diluted in Tris-yolk extender supplemented with vitamin C (2.5mmol/mL); glutathione group-semen diluted in Tris-yolk extender supplemented with reduced glutathione (2.5mmol/mL) and associated group-semen diluted in Tris-yolk extender supplemented with vitamin C (1.25mmol/mL) and reduced glutathione (1.25mmol/mL). Afterwards, the semen was packed into French straws and submitted to cryopreservation using automated equipment. After cryopreservation, the semen was thawed and evaluated considering sperm motility, morphology, plasma membrane, acrosome, mitochondrial potential and oxidative stress, as well as the thermo resistance test. Extender’s supplementation with the association of vitamin C and reduced glutathione showed benefic effects on sperm motility and preservation of plasma and acrosomal membranes during semen cryopreservation, being also the group that showed higher values of reactive oxygen species. Thus, the association of both antioxidants contributed to the preservation of sperm cells in every analyzed characteristic, suggesting its use on bovine semen cryopreservation.

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Impact of supplementing commercial rabbit pellets with fresh Moringa oleifera leaves on semen characteristics in rabbit bucks under tropical climate in Trinidad

Caribbean Medical Journal ◽

10.48107/cmj.2021.04.001 ◽

2021 ◽

Author(s):

Krishna Mohan Kumar

Keyword(s):

Sperm Motility ◽

Moringa Oleifera ◽

Sperm Count ◽

Control Group ◽

Computer Assisted ◽

Group Iii ◽

Group I ◽

Significant Difference ◽

Moringa Oleifera Leaves ◽

The Impact

Objective This study aimed to evaluate the impact of the dietary supplement of Moringa oleifera leaves (MOL) on semen quality and characteristics in rabbits. Methods Eighteen (n=18) breeding bucks of New Zealand white, of similar age group, were used for the study. Three feeding regimes, (i) 100% commercial rabbit pellets (CRP)-Group I (ii) 90% CRP + 10% fresh MOL on a dry matter (DM) basis – Group II and (iii) 80% CRP + 20% fresh MOL on a DM basis – Group III, were adopted and the trial continued for 21 days. After adaptation to the diet, semen was collected from each buck and subjected to evaluation using a computer-assisted semen analyser. Results In Group III, the sperm count, normal sperm morphology, and sperm motility increased (52.0%) in comparison with the control (Group I; 50.1%). The inclusion of 20% Moringa oliefera in the diet (Group III) caused a significant increase (P<0.05) in semen concentration (Control =136.2 M/mL; Group III=297.2 M/mL). There was no significant difference (P>0.05) in sperm motility and semen volume among the groups. Conclusion The results suggest that supplementing commercial rabbit pellets with 20% fresh Moringa oliefera leaves on a DM basis can improve the quality and characteristics of semen in breeding bucks.

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Effect of Tramadol on Sperm Profile of Male Albino Rats

Asian Journal of Research in Biochemistry ◽

10.9734/ajrb/2018/v3i429844 ◽

2019 ◽

pp. 1-6

Author(s):

I. S. Esua ◽

U. U. Uno ◽

U. B. Ekaluo

Keyword(s):

Sperm Motility ◽

Sperm Count ◽

Sperm Head ◽

Male Rats ◽

Control Group ◽

Albino Rats ◽

Dependent Manner ◽

Sperm Viability ◽

Albino Rat ◽

Significant Difference

Background and Aim: Tramadol is a potent analgesic effective in the treatment of mild to severe pains. However, the use of the drug can pose a threat to other organs and systems. Therefore, this study evaluated the effect of graded doses of tramadol on sperm profile of male albino rats. Materials and Methods: Eighteen male rats were divided into three groups (A, B and C) using completely randomized design (CRD) with six rats in each group. Rats in group A served as the control group and were given just food and water while groups B and C were given tramadol at 50 and 100 mg/kg body weight (BW) respectively, daily for the period of 65 days. The treatment was administered via oral gavage and at the end of the treatments, the rats were sacrificed. Immediately after sacrifice, a puncture was made in the epididymis with a sterile pin and examined for semen pH. The epididymes were processed for epididymal sperm motility, viability, count and sperm head abnormality. Results: There was no significant difference in the weight of testes and semen pH. Sperm viability, sperm motility, sperm count and weight of epididymes significantly reduced (p<0.05) in tramadol treated animals when compared with the control. Results also indicated statistically significant (p<0.05) increase in sperm head abnormalities in rats treated with tramadol when compared with the control. Conclusion: The results obtained from this study reveal that tramadol has negative effects on weight of epididymes, sperm count, sperm viability, sperm motility and sperm head abnormalities in male albino rat as mammalian models in a dose dependent manner.

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Antioxidant effect on viability of boar semen cooled to 15°C

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-11294 ◽

2020 ◽

Vol 72 (1) ◽

pp. 145-152

Author(s):

V.E.D. Martins ◽

S.C.C. Pinto ◽

R.M. Chaves ◽

A.K.D. Barros Filho ◽

L.M. Laskoski ◽

...

Keyword(s):

Vitamin C ◽

Sperm Motility ◽

Reduced Glutathione ◽

Antioxidant Effect ◽

Control Group ◽

Promising Alternative ◽

Sperm Characteristics ◽

Boar Semen

ABSTRACT This study aimed to evaluate the addition of Vitamin C, reduced Glutathione and trolox on sperm characteristics of pork refrigerated semen. Six pigs were collected through the technique of gloved hand (10 ejaculates/animals). The semen was diluted in MR-A®. After the previous evaluations, the treatments were added: Control group: diluent only; Vitamin C Group: 200μM/mL Vitamin C; Trolox Group: 200μM/mL Trolox; Glutathione group: 2.5mM/ml Reduced glutathione. The semen was stored in thermal boxes and placed inside the refrigerator at 15oC and evaluated at D0, 12, 48, 72 hours. After 30 hours of incubation, each treatment was divided into two equal fractions and the same concentration of antioxidants was added in one of the parts. The results show that reduced glutathione supplementation preserves sperm motility after 24 hours but also has a higher percentage of acrosome intact in the presence of this antioxidant. There was no effect of adding a second dose of the antioxidants. In conclusion, the addition of reduced Glutathione to the swine semen diluent is a promising alternative for better preservation of sperm characteristics and the addition of the second dose of antioxidants during storage is detrimental to semen.

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Experimental diet based on the foods listed in the Family Budget Survey is more detrimental to growth than to the reflex development of rats

Revista de Nutrição ◽

10.1590/s1415-52732013000200006 ◽

2013 ◽

Vol 26 (2) ◽

pp. 177-196 ◽

Cited By ~ 6

Author(s):

Michelle Figueiredo Carvalho ◽

Meury Kétteryn Monteiro Evangelista da Costa ◽

Gisélia de Santana Muniz ◽

Raul Manhães de Castro ◽

Elizabeth do Nascimento

Keyword(s):

Physical Growth ◽

Control Group ◽

Free Access ◽

Lactation Period ◽

Experimental Diet ◽

Entire Study ◽

The Family ◽

Pregnancy And Lactation ◽

Reflex Control ◽

The Impact

OBJECTIVE: The present study assessed the pregnancy and lactation performances of rats fed an experimental diet based on the foods listed in the Family Budget Survey (Pesquisa de Orçamento Familiar) 2002/2003 and the impact of said diet on the growth and development of the pups until weaning. METHODS: Wistar (n=12) rats were randomly divided into two groups: a control group (control group, n=6) fed a commercial chow (Labina®, Brazil) and an experimental group (n=6) fed the Family Budget Survey diet during the entire pregnancy and lactation period. All animals had free access to food and water during the entire study period. RESULTS: The Family Budget Survey diet increased the duration of pregnancy (control group=21.00±0.00; POFG=21.57±0.55, p=0.025) and made the dams lose weight during the lactation period (control group=27.92±18.47g; POFG=-15.66±16.90g). The Family Budget Survey group presented low food, energy and nutrient intakes during pregnancy, which became even lower during lactation. Pups from Family Budget Survey dams presented lower body weight at weaning (control group=52.38±4.49g; POFG=39.88±2.78g, p=0.001) and lower nose-to-anus length (control group= 117.37±0.64mm; POFG=125.62±0.96mm, p=0.001). However, some physical milestones and reflexes occurred earlier, such as the placing response reflex [control group= 12.00 (9.00-15.00) days; POFG=9.50 (9.00-14.00) days] aerial righting reflex [control group=18.00 (17.00-20.00) days; POFG=16.00 (13.00-18.00) days] and unfolding of the external ear [control group=3.00 (3.00-3.00) days; POFG=2.00 (2.00-3.00) days]. CONCLUSION: The Family Budget Survey diet seems to be more detrimental to the physical growth of the pups than to their brain growth, according to the assessed reflexes and physical milestones and measures. This may be due to the low protein content of the diet for rat reproduction and growth combined with adequate fat and essential fatty acid contents. Providing an adequate amount of protein to the Family Budget Survey diet may normalize pup growth.

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Positive effects of trehalose and cysteine on ram sperm parameters

Veterinární Medicína ◽

10.17221/131/2016-vetmed ◽

2017 ◽

Vol 62 (No. 5) ◽

pp. 245-252 ◽

Cited By ~ 2

Author(s):

S. Gungor ◽

C. Ozturk ◽

AD Omur

Keyword(s):

Sperm Motility ◽

Sperm Parameters ◽

Control Group ◽

Mitochondrial Activity ◽

Sperm Viability ◽

Significant Protection ◽

Positive Effects ◽

Liquid Storage ◽

Acrosome Integrity ◽

Ram Sperm

The aim of this study was to determine the effects of trehalose and cysteine on sperm motility, viability, mitochondrial activity and acrosome integrity during liquid storage of Merino ram semen. Ejaculates were collected using artificial vaginas from five Merino rams, microscopically evaluated and pooled at 37 °C. The pooled semen samples were diluted in a Tris-based extender, including cysteine (2 mM and 4 mM), trehalose (10 mM and 25 mM) and no antioxidant (control). Diluted semen samples were kept in tubes and cooled from 37 to 5 °C in a cold cabinet, and maintained at 5 °C. Cooled samples were evaluated for sperm motility, viability, mitochondrial activity and acrosome integrity at 0, 24, 48, 72 and 96 h. Extender supplemented with trehalose (10 and 25 mM) and cysteine (2 and 4 mM) led to higher motility in comparison to the control at 24, 48, 72 and 96 h of liquid storage (P < 0.05). Trehalose at the doses of 10 mM, 25 mM and 2 mM cysteine led to higher viability between 24–48–72 h and at 96 h of liquid storage (P < 0.05). Further, 4 mM of cysteine improved sperm viability rates at 24 and 48 h of storage compared to the control group (P < 0.05), and resulted in improved acrosome integrity rates compared to the control group at 72 and 96 h of storage (P < 0.05). Extender supplemented with 10 and 25 mM trehalose at 24 and 72 h and 4 mM cysteine at 24 and 96 h of storage led to higher sperm mitochondrial activity rates when compared to the control group (P < 0.05). In conclusion, the findings of this study show that trehalose and cysteine provided significant protection to ram sperm parameters during liquid storage.

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Ascorbic Acid, Inflammatory Cytokines (IL-1β/TNF-α/IFN-γ), or Their Combination’s Effect on Stemness, Proliferation, and Differentiation of Gingival Mesenchymal Stem/Progenitor Cells

Stem Cells International ◽

10.1155/2020/8897138 ◽

2020 ◽

Vol 2020 ◽

pp. 1-14

Author(s):

Karim M. Fawzy El-Sayed ◽

Nhung Nguyen ◽

Christof E. Dörfer

Keyword(s):

Ascorbic Acid ◽

Progenitor Cells ◽

Inflammatory Cytokines ◽

Cellular Proliferation ◽

Basic Medium ◽

Control Group ◽

Multilineage Differentiation ◽

Positive Effects ◽

Proliferation And Differentiation ◽

The Impact

Objective. Ascorbic acid (AA) and controlled inflammatory stimuli are postulated to possess the ability to independently exert positive effects on a variety of proliferative, pluripotency, and differentiation attributes of gingival mesenchymal stem/progenitor cells (G-MSCs). The current study’s objective was to explore and compare for the first time the impact of the major inflammatory cytokines (IL-1β/TNF-α/IFN-γ), AA, or their combination on multipotency/pluripotency, proliferative, and differentiation characteristics of G-MSCs. Design. Human G-MSCs (n=5) were isolated and cultured in basic medium (control group), in basic medium with major inflammatory cytokines; 1 ng/ml IL-1β, 10 ng/ml TNF-α, and 100 ng/ml IFN-γ (inflammatory group), in basic medium with 250 μmol/l AA (AA group) and in inflammatory medium supplemented by AA (inflammatory/AA group). All media were renewed three times per week. In stimulated G-MSCs intracellular β-catenin at 1 hour, pluripotency gene expression at 1, 3, and 5 days, as well as colony-forming units (CFUs) ability and cellular proliferation over 14 days were examined. Following a five-days stimulation in the designated groups, multilineage differentiation was assessed via qualitative and quantitative histochemistry as well as mRNA expression. Results. β-Catenin significantly decreased intracellularly in all experimental groups (p=0.002, Friedman). AA group exhibited significantly higher cellular counts on days 3, 6, 7, and 13 (p<0.05) and the highest CFUs at 14 days [median-CFUs (Q25/Q75); 40 (15/50), p=0.043]. Significantly higher Nanog expression was noted in AA group [median gene-copies/PGK1 (Q25/Q75); 0.0006 (0.0002/0.0007), p<0.01, Wilcoxon-signed-rank]. Significant multilineage differentiation abilities, especially into osteogenic and chondrogenic directions, were further evident in the AA group. Conclusions. AA stimulation enhances G-MSCs’ stemness, proliferation, and differentiation properties, effects which are associated with a Wnt/β-catenin signaling pathway activation. Apart from initially boosting cellular metabolism as well as Sox2 and Oct4A pluripotency marker expression, inflammation appeared to attenuate these AA-induced positive effects. Current results reveal that for AA to exert its beneficial effects on G-MSCs’ cellular attributes, it requires to act in an inflammation-free microenvironment.

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Postoperative Glycaemia in Patients Following the CABG Surgery

Bosnian Journal of Basic Medical Sciences ◽

10.17305/bjbms.2008.2997 ◽

2008 ◽

Vol 8 (1) ◽

pp. 48-51

Author(s):

Mirsad Kacila ◽

Elnur Tahirović ◽

Zina Lazović

Keyword(s):

Artery Bypass ◽

Medical Documentation ◽

Control Group ◽

Entire Study ◽

Antidiabetic Therapy ◽

Glucose Levels ◽

Study Results ◽

Before And After ◽

Glycaemia Control ◽

The Impact

In this study we analyzed patients glucose levels taken before and after coronary artery bypass surgery. The data are taken from University of Sarajevo Clinics Centre-medical documentation of Heart Center from January 1st, 2007 to October 31st, 2007. Therefore, the entire study is done retrospectively. The patients considered in this analysis were divided into three groups. The patients in the first group were treated with peroral antidiabetics. The second group included patients treated with insulin while the third group consisted of patients without diabetes and acted as control group. All the patients had increased BMI. In the first two groups glucose levels were elevated before the surgery. Data analysis showed that three patients from the first group changed from peroral antidiabetics to insulin during postoperative period, and they were dismissed from hospital with this therapy. In the control group, peroral antidiabetis were introduced in four patients. The study results show that all the three groups have had high glycaemia on the first postoperative day. This may be caused by a number of reasons: inadequate pre-operative antidiabetic therapy, the impact of stress during surgery, inadequate pre-operative treatment of glycaemia or avoidance of the proscribed therapy by patients themselves. Stress during operation and administration of several medications after operation may also cause increased insulin resistance and distort glycaemia control.

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Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm

Veterinary Sciences ◽

10.3390/vetsci8120302 ◽

2021 ◽

Vol 8 (12) ◽

pp. 302

Author(s):

Iván Yánez-Ortiz ◽

Jaime Catalán ◽

Ariadna Delgado-Bermúdez ◽

Augusto Carluccio ◽

Jordi Miró ◽

...

Keyword(s):

Reduced Glutathione ◽

Membrane Integrity ◽

Redox Balance ◽

Low Fertility ◽

Ros Generation ◽

Enzymatic Antioxidants ◽

Freezing Medium ◽

Exogenous Addition ◽

Sperm Plasma Membrane ◽

The Impact

In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O2−) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.

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Evaluation of the Dietary Supplementation of a Formulation Containing Ascorbic Acid and a Solid Dispersion of Curcumin with Boric Acid against Salmonella Enteritidis and Necrotic Enteritis in Broiler Chickens

Animals ◽

10.3390/ani9040184 ◽

2019 ◽

Vol 9 (4) ◽

pp. 184 ◽

Cited By ~ 5

Author(s):

Daniel Hernandez-Patlan ◽

Bruno Solís-Cruz ◽

Karine Patrin Pontin ◽

Juan D. Latorre ◽

Mikayla F. A. Baxter ◽

...

Keyword(s):

Ascorbic Acid ◽

Boric Acid ◽

Solid Dispersion ◽

Broiler Chickens ◽

Salmonella Enteritidis ◽

Control Group ◽

Necrotic Enteritis ◽

The Impact ◽

Therapeutic Administration ◽

Intestinal Iga

Two experiments were conducted to evaluate the effect of the prophylactic or therapeutic administration of a 0.1% mixture containing ascorbic acid and a solid dispersion of curcumin with polyvinylpyrrolidone and boric acid (AA-CUR/PVP-BA) against Salmonella Enteritidis (S. Enteritidis) in broiler chickens. A third experiment was conducted to evaluate the impact of the dietary administration of 0.1% AA-CUR/PVP-BA in a necrotic enteritis (NE) model in broiler chickens. The prophylactic administration of 0.1% AA-CUR/PVP-BA significantly decreased S. Enteritidis colonization in cecal tonsils (CT) when compared to the positive control group (PC, p < 0.05). The therapeutic administration of 0.1% AA-CUR/PVP-BA significantly reduced the concentration of S. Enteritidis by 2.05 and 2.71 log in crop and CT, respectively, when compared with the PC on day 10 post-S. Enteritidis challenge. Furthermore, the serum FITC-d concentration and total intestinal IgA levels were also significantly lower in chickens that received 0.1% AA-CUR/PVP-BA. Contrary, the PC group showed significantly higher total intestinal IgA levels compared to the negative control or AA-CUR/PVP-BA groups in the NE model. However, 0.1% AA-CUR/PVP-BA showed a better effect in reducing the concentration of S. Enteritidis when compared to the NE model. Further studies with higher concentration of AA-CUR/PVP-BA into the feed to extend these preliminary results are currently being evaluated.

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