107 MOTILITY PARAMETER PATTERNS OF NGUNI BULLS: EFFECT OF VARIOUS GYCEROL CONCENTRATIONS FOLLOWING CRYOPRESERVATION

2010 ◽  
Vol 22 (1) ◽  
pp. 212
Author(s):  
T. L. Nedambale ◽  
M. L. Mphaphathi ◽  
P. H. Munyai ◽  
M. Tshabalala ◽  
P. Malusi ◽  
...  
Keyword(s):  
South Africa ◽  
South African ◽  
Semen Analysis ◽  
Egg Yolk ◽  
Glycerol Concentration ◽  
Department Of Agriculture ◽  
Spermatozoa Motility ◽  
Bull Semen ◽  
Significant Difference ◽  
Motility Parameters

The Nguni breed of South Africa is small, hardy, disease-tolerant, thrives on poor pasture, and was regarded as an inferior breed in the past. For optimizing routine fresh and freezing of Nguni bull semen analysis, 3 different concentrations of glycerol (7, 10, and 14%) were examined. Ten ejaculates were collected from each 6 Nguni bulls using electro-ejaculator at ARC, Irene, South Africa. Following semen collection, semen was examined for macroscopic (volume, pH, and concentration) and microscopic (motility) parameters. The semen was extended with Tris + 10% egg yolk diluent at a ratio of 1 : 2 (v/v) and frozen at different concentrations of glycerol (7, 10, and 14%). The semen was then evaluated using the sperm class analyzer (SCA; CASA system) for progressive motility parameters. Fresh and frozen-thawed were fixed and stained with Nigrosin-Eosin for morphology (dead and live). Data were analyzed by ANOVA. There was a significant difference among individual Nguni bull spermatozoa volume and concentration. Analyzed frozen-thawed Nguni spermatozoa resulted in a significant (P < 0.05) difference of spermatozoa motility parameters frozen in 10% glycerol (68%) compared with 7 (41%) and 14% glycerol (30%). In conclusion, Nguni spermatozoa can be cryopreserved successfully when 10% of glycerol concentration is used. The results of this study will improve the viability of cryopreserved Nguni bull spermatozoa following the development of a South African semen cryo-gene bank. This study was supported by grants from National Research Foundation (NRF), Hungarian, South African Bilateral Scientific and Technological (TETNo. OMFB-00302/2008, RT24000) collaborative project. Department of Agriculture Forestry and Fisheries (DAFF, RPPP15).

scholarly journals Antioxidant Potential of Ferulic Acid on the Freezability of Bull Semen

2019 ◽  
Vol 4 (3) ◽  
pp. 1-4
Author(s):  
Omur AD
Keyword(s):  
Ferulic Acid ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Single Step ◽  
Spermatozoa Motility ◽  
Bull Semen ◽  
Sperm Density ◽  
Significant Difference ◽  
Artificial Vagina

Ejaculates were collected twice a week from the bulls, via an artificial vagina, during two weeks. The suitable ejaculates obtained for sperm density (≥ 1.4 × 10 9 spermatoz oa / ml) and for motility (≥ 75%) were used for dilution and freezing of semen. A Tris - based extender (Tris 297.58mM, citric acid 96.32mM, fructose 82.66mM, egg yolk 15% (v/v), glycerol 5% (v/v), gentamicin 0.1 ml / 100ml, pH 6.8 - 7.0) was used as the base extender (cryopreservation diluent). Pooled ejaculate was split into 2 equal aliquots and diluted at 32 °C with base extender containing ferulic acid (100 μM) and no antioxidant (control), respectively. Each aliquot was diluted to a final semen concentrati on of approximately 1.2 × 10 8 sperm/ml (single step dilution), in 15 - ml polypropylene centrifuge tubes. After dilution, semen samples were kept at room temperature for 10 minutes then, the diluted semen samples were aspirated into 0.25 ml French straws, seal ed with polyvinyl alcohol powder and equilibrated at 5 °C for 3 h. After equilibration, the straws were frozen in liquid nitrogen vapour (4 cm above the liquid nitrogen, ~ - 100 o C ) for 10 min and then plunged into liquid nitrogen for storage, - 196 o C . In the study, sperm samples containing antioxidant and non - antioxidant were evaluated for spermatozoa motility and membrane integrity after freezing / thawing. In the present study, no statistically significant difference was found between the control and experim ental groups for motility and membrane integrity after freeze - thawing. The application consisted of 4 replications.

76 THE EFFECT OF GLYCEROL CONCENTRATIONS IN EGG-YOLK CITRATE EXTENDER ON THE QUALITY OF CRYOPRESERVED NGUNI BULL SEMEN

2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
F. V. Ramukhithi ◽  
T. R. Netshirovha ◽  
C. Hlungwani ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Rural Areas ◽  
Egg Yolk ◽  
Positive Control ◽  
Negative Control ◽  
Glycerol Concentration ◽  
Bull Semen ◽  
Frozen Semen ◽  
Thawing Process ◽  
Motility Rate

There are bull shortages in South African poor rural areas. Artificial-insemination technology could play a significant role on breeding emerging farmer’s cattle. The objective of this study was to compare glycerol concentrations (0, 4, 8, or 12%) during freezing of Nguni bull semen to conduct AI in different villages. Semen was collected by electro ejaculator from 2 Nguni bulls of known and proven fertility. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories within 10 min after collections. Semen samples were pooled and evaluated by Sperm Class Analyser® and allocated randomly per treatment. Semen was then diluted (1 : 2 v:v) with egg-yolk citrate extender supplemented with either 0% (negative control), 4, 8, and 12% of glycerol concentration or AndroMed® (positive control). Semen samples were equilibrated for 4 h at 5°C. After equilibration period, samples were loaded into 0.5-mL straws and placed horizontally into the controlled rate (–5, –8, –10, –12, –15, –25, –35°C min–1) from 5°C until target temperature of –80°C is reached. The frozen semen straws were stored in a liquid nitrogen tank (–196°C) until thawing. Treatment means were separated using Fisher’s protected t-test least, and data are presented as mean ± SD. There was a significant differences (P < 0.05) between raw total sperm motility (83.3 ± 19.3) and frozen–thawed sperm with either 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), or 12% (61.5 ± 4.7) of glycerol and on AndroMed® (27.7 ± 17.8) group. Regardless of the glycerol concentrations used, the freezing-thawing process reduced (P < 0.05) the Nguni total sperm motility rate compared to uncryopreserved sperm (83.3 ± 19.3). In conclusion, egg-yolk citrate extender supplemented with 12% glycerol yielded a better (P < 0.05) total sperm motility rate (61.5 ± 4.7) as compared with the 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), and AndroMed® (27.7 ± 17.8) group. Further studies are required to test other levels of glycerol concentrations (>12%) on freezing Nguni semen and conducting AI.

71 COMPARISON OF CRYOPROTECTANTS ON CRYOPRESERVATION OF VENDA COCK SPERMATOZOA

2009 ◽  
Vol 21 (1) ◽  
pp. 135
Author(s):  
M. L. Mphaphathi ◽  
M. B. Raito ◽  
M. B. Makhafola ◽  
D. Luseba ◽  
T. L. Nedambale
Keyword(s):  
South Africa ◽  
Ethylene Glycol ◽  
South African ◽  
Sperm Quality ◽  
Target Temperature ◽  
Department Of Agriculture ◽  
Semen Collection ◽  
Frozen Semen ◽  
Contrast Microscopy ◽  
Motility Rate

Improving the cryopreservation technique for indigenous fowl semen may contribute to the development of cryogene banks in South Africa. The goal was to identify a cryoprotectant among dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propanediol (PND) that is compatible with survival after freezing of Venda cock spermatozoa. Six Venda cocks were used for semen collection. The abdominal massaging technique was applied for semen collection from cocks. Individual ejaculates were diluted with modified Kobidil+ (mK+) extender (extender A) at ratio of 1:2 (v/v) before freezing, and equilibrated for 2 h. Semen was diluted again at a ratio of 1:1 (v/v) with mK+ plus 8% DMSO, EG, and PND (extender B) and equilibrated for 2 h at 5°C. Semen were then transferred into 0.25-mL plastic straws and placed into a programmable freezer (Planer Kryosave). The temperature of the chamber was decreased in a stepwise manner, from 5°C at a rate of 1°C min–1 until it reached the target temperature of –20°C. Finally, the straws were exposed to liquid nitrogen (LN2) vapor and plunged into LN2 (–196°C). The semen straws were stored in an LN2 tank at –196°C. After 1 week, frozen semen straws were thawed at 5°C for evaluation of spermatozoa survivability and motility rate at 0, 30, 60, and 90 min, using contrast microscopy (20× magnification). Data were analyzed by ANOVA. Spermatozoa live and motility rates were greater before freezing (Table 1) in all groups. There was no significance difference between DMSO and EG with regard to live and motility rates. However, the lowest rates of live and motility spermatozoa were recorded in the PND group. In conclusion, this study demonstrated that the cryopreservation process reduces sperm quality and propanediol was not suitable for cryopreserving Venda cock spermatozoa. Table 1.Comparison of three CPA on cryopreservation of Venda cock semen This study was funded by the South African National Department of Agriculture, ARC, DST-PDP (RT19000), and National Research Foundation (NRF, Grant. no. RT21 and 24000).

scholarly journals 12 CASA PARAMETERS OF FRESH BULL SEMEN COLLECTED BY ARTIFICIAL VAGINA OR ELECTROEJACULATION IN ARGENTINA

2005 ◽  
Vol 17 (2) ◽  
pp. 156 ◽  
Author(s):  
G. Brogliatti ◽  
F. Garcia Migliaro ◽  
R. Cavia ◽  
G. Larraburu ◽  
A. Albrecht
Keyword(s):  
Semen Analysis ◽  
Glass Tube ◽  
Computer Assisted ◽  
Bull Semen ◽  
Straight Line ◽  
Beef Bulls ◽  
Semen Extender ◽  
Artificial Vagina ◽  
Lateral Head ◽  
Motility Parameters

The latest entry in the field of semen evaluation is computer assisted semen analysis (CASA). Its greatest advantages are elimination of the subjective nature of routine semen evaluation and the addition of detailed motion analysis unquantifiable by visual examination. The objective of this study was to evaluate CASA motility parameters of fresh bull semen collected by artificial vagina (AV) or electroejaculation (EE) from a total of 56 beef different bulls. Semen samples from a total of 45 beef bulls were collected by AV from winter to the end of spring (740 collections), and from 11 beef bulls by EE (120 collections) in the same period. First and second AV collections were analyzed as individual data. EE collection was performed only one. Means and standard deviations for each characteristic were calculated, compared, and statistically analyzed. A sample of the collection was diluted 1:20 in a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) and held in a glass tube at 36°C for 5 min before analysis. The sample was loaded into 20-μm chambers, and six microscope fields from each chamber were analyzed. The following sperm motility parameters were determined with the Ceros 12.1 sperm analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) on at least 1000 spermatozoa:concentration (CONC), velocity average path (VAP), velocity straight line (VSL), curvilinear velocity (VCL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of rapid or statics cells. There were no significant differences (P > 0.05) in VAP, VSL, VCL, ALH, STR, LIN, and the percentage of rapid and static cells of semen collected by AV or EE. The concentration (sperm/mL) of the AV-collected sperm was significantly higher than for the sperm collected by EE. Results from the analysis indicate that semen collected by artificial vagina have motility characteristics similar to those collected by electroejaculation. More research needs to be done to evaluate motility parameters of frozen/thawed semen collected by electroejaculation and by artificial vagina. This research was supported by Centro Genético Bovino de EOLIA sa Argentina.

scholarly journals The development and investigation of the psychometric properties of a burnout scale within a South African agricultural research institution

2014 ◽  
Vol 40 (1) ◽  
Author(s):  
Doris N. Asiwe ◽  
Lené I. Jorgensen ◽  
Carin Hill
Keyword(s):  
South Africa ◽  
South African ◽  
Quantitative Research ◽  
Agricultural Research ◽  
Cross Sectional Survey ◽  
Cross Sectional ◽  
Research Institution ◽  
Significant Difference ◽  
New Instrument ◽  
Construct Equivalence

Orientation: Burnout of employees is well documented within South Africa, but researchers have adapted imported instruments with a number of limitations. Therefore there is a need to develop a new instrument suitable for use in South Africa.Research purpose: To give an overview of current burnout measures, identify gaps within the literature and develop a new burnout scale for use within South Africa. The research examined the construct validity, reliability, construct equivalence and item bias of this new scale and investigated any differences that exist in relation to demographic variables.Motivation for the study: This study aimed to address various limitations regarding existing measures by developing a reliable and valid instrument for measuring burnout in South African employees that includes cognitive, physical and emotional (affective) components.Research approach, design and method: This empirical, quantitative research study delivered a cross-sectional survey, including the burnout scale and a biographical data questionnaire, to 443 employees of an agricultural research institution. Items for the burnout scale were written based on a literature review.Main findings: Exploratory factor analysis with target rotations resulted in a three-factor burnout model. Reliability analysis showed that all three scales (1) were sufficiently internally consistent and (2) showed construct equivalence for Black and White employees and speakers of Afrikaans and African languages. A practically significant difference in burnout levels was found in relation to age.Practical/managerial implications: The scale can be used to assess burnout for different cultural groups within research-based institutions.Contribution/value-add: This study contributes to knowledge regarding the burnout levels of employees in an agricultural research institution in South Africa and provides a new burnout scale that can be utilised in similar institutions.

scholarly journals Naturally acquired antibodies to Bacillus anthracis protective antigen in vultures of southern Africa

2008 ◽  
Vol 75 (2) ◽  
Author(s):  
P. C.B. Turnbull ◽  
M. Diekman ◽  
J. W. Killian ◽  
W. Versfeld ◽  
V. De Vos ◽  
...  
Keyword(s):  
South Africa ◽  
Bacillus Anthracis ◽  
South African ◽  
Southern Africa ◽  
Protective Antigen ◽  
Control Group ◽  
North West ◽  
Age Related ◽  
Significant Difference ◽  
And Control

TURNBULLP, P.C.B. DIEKMANNM,M., KILIAN, J.W., VERSFELDW, W.,DE VOS, V., ARNTZENL, L.,WOLTER, K., BARTELS, P. & KOTZE, A. 2008.N aturally acquired antibodies to Bacillusa nthracisp rotective antigeni n vultureso f southern Africa. Onderstepoort Journal of Veterinary Research, T5:95-102 Sera from 19 wild caught vultures in northern Namibia and 15 (12 wild caught and three captive bred but with minimal histories) in North West Province, South Africa, were examined by an enzyme-linked immunosorbenats say( ELISA)f or antibodiesto the Bacillus anthracis toxin protective antigen (PA). As assessed from the baseline established with a control group of ten captive reared vultures with well-documented histories, elevated titres were found in 12 of the 19 (63%) wild caught Namibian birds as compared with none of the 15 South African ones. There was a highly significant difference between the Namibian group as a hole and the other groups (P < 0.001) and no significant difference between the South African and control groups (P > 0.05). Numbers in the Namibian group were too small to determine any significances in species-, sex- or age-related differences within the raw data showing elevated titres in four out of six Cape Vultures, Gyps coprotheress, six out of ten Whitebacked Vultures, Gyps africanus, and one out of three Lappet-faced Vultures, Aegypiust racheliotus, or in five of six males versus three of seven females, and ten of 15 adults versus one of four juveniles. The results are in line with the available data on the incidence of anthrax in northern Namibia and South Africa and the likely contact of the vultures tested with anthrax carcasses. lt is not known whether elevated titre indicates infection per se in vultures or absorption of incompletely digested epitopes of the toxin or both. The results are discussed in relation to distances travelled by vultures as determined by new tracking techniques, how serology can reveal anthrax activity in an area and the issue of the role of vultures in transmission of anthrax.

scholarly journals KUALITAS MOTILITAS DAN VIABILITAS SPERMATOZOA DARI SEMEN AFKIR SAPI LIMOUSIN PADA PENGENCER SUSU SKIM KUNING TELUR SITRAT DENGAN PENAMBAHAN BERBAGAI KADAR GLUKOSA

2020 ◽  
Vol 7 (2) ◽  
pp. 96
Author(s):  
Rudi Irvanto ◽  
Hardijanto Hardijanto ◽  
Widya Paramita ◽  
Suherni Susilowati ◽  
Tita Damayanti L ◽  
...  
Keyword(s):  
Research Design ◽  
Glucose Level ◽  
Skim Milk ◽  
Egg Yolk ◽  
Spermatozoa Motility ◽  
Bull Semen ◽  
Randomized Design ◽  
Completely Randomized Design

Quality of spermatozoa motility and viability from rejected limousin bull semen diluted with skim milk egg yolk sitrat added with various levels of glucose. Glucose level used were 0%, 0,5%, 1,5%, 2,5%, and 3,5%. Writer was using on four years old Limousine bull. Bull semen used in this research was bull rejected semen with bellow 70% motility. Semen observation was done at 0 hour, 24 hours and 48 hours. Research design used in this study was completely randomized design with faktorial pattern with 5 replicates. Highest result in motility this research was showed at 24 hours with 30% value in glucose 2,5% treatment and 48 hours with 10% value. The lowest result showed in glucose 0% treatment at 24 hours and 0% at 48 hours. Highest result in viability showed on glucose 2,5% treatment with 62,6% value at 24 hours and at 48 hours with 53,4% value. Lowest result in viability showed on glucose 0% treatment with 44,2% value at 24 hours and 31,4% value at 48 hours.

53 FREEZING BULL SEMEN IN A SYNTHETIC MEDIUM

2017 ◽  
Vol 29 (1) ◽  
pp. 134
Author(s):  
L. Gavin-Plagne ◽  
P. Bodranghien ◽  
A. Vachet ◽  
L. Commin ◽  
S. Buff ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Synthetic Medium ◽  
Calf Serum ◽  
Egg Yolk ◽  
Wilcoxon Test ◽  
Computer Assisted ◽  
Sperm Cells ◽  
Bull Semen ◽  
Significant Difference

Animal-derived products are currently used to cryopreserve sperm cells. However, these products represent potential risks of contamination by pathogens. Optidyl® (IMV Technologies, L’Aigle, France), containing egg yolk, is a reference product in Europe used routinely in bovine insemination centers. Commercial media such as soy lecithin or liposome-based media have been used to replace extenders containing products derived from animals. However, their protective effect could be called into question because of their non-synthetic or unstable properties. Despite these innovative extenders on the market, it might be necessary for sanitary reasons to cryopreserve bull semen in a stable and synthetic extender. CRYO3 (Stem Alpha, Saint-Genis l’Argentière, France), a serum-free and protein-free medium used for cryopreserving somatic and stem human cells, is a potential medium to cryopreserve reproductive cells. Recently, CRYO3 improved cryopreservation of in vitro-produced bovine embryos compared with fetal calf serum and BSA-based media. Thus, it could be interesting to test this medium on sperm cells. The objective of this study was to compare 2 in vitro freezing media on bull semen: a commercial egg yolk-based medium and a synthetic medium containing 20% CRYO3. Sperm from 5 bulls were collected in Auriva station (Brindas, France). A sample of each ejaculate was used to assess fresh semen quality. The remaining sperm of each bull was split and diluted in 2 media: Optidyl® and a CRYO3-based medium. Semen was equilibrated at least 4 h at 4°C before being packaged in 0.25-mL French straws, and then frozen into a programmable freezer and plunged into liquid nitrogen. Osmolarity and pH were respectively 1462 mOsm/kg and 6.9 for Optidyl® and 1286 mOsm/kg and 6.8 for CRYO3 medium. Viability (with SYBR-14 and propidium iodide) and high mitochondrial membrane potential (hMMP) (with JC-1) were assessed using flow cytometry. A hypo-osmotic swelling test was performed to assess functional membrane integrity (FMI). The motility parameters were evaluated by computer-assisted sperm analysis. Statistical analysis was performed using Wilcoxon test with the R software. Fresh sperm showed 52% viability, 64% hMMP, 74% FMI, and 62 and 76% progressive and total motility, respectively. For all parameters measured, no significant difference was observed between extenders and between fresh and frozen–thawed sperm (P > 0.05). However, Optidyl® showed clearly better survival than CRYO3 (45% v. 16% for viability, 58% v. 20% for hMMP, 67% v. 25% for FMI, 51% v. 14% for progressive motility and 72% v. 32% for total motility, respectively). These results show that it is possible to freeze bovine semen in synthetic extender though the low survival rate after freezing-thawing. Indeed, it is known that motility and flow cytometry parameters are not necessarily good indicators of fertility. Artificial inseminations will be done to verify the fertility of the sperm cryopreserved in CRYO3. Repetitions with more bulls from different breeds will be performed to complete this preliminary work. This work was supported by grant CRB-ANIM ANR-11-INBS-0003.

scholarly journals 7 HETEROSPERMIC INSEMINATION AT TWO SPERM CONCENTRATIONS IN TIMED AI: CASA SEMEN PARAMETERS AND PREGNANCY RATES

2005 ◽  
Vol 17 (2) ◽  
pp. 154 ◽  
Author(s):  
A. Albrecht ◽  
R. Cavia ◽  
G. Larraburu ◽  
F. Garcia Migliaro ◽  
G. Brogliatti
Keyword(s):  
Artificial Insemination ◽  
Semen Analysis ◽  
Pregnancy Rates ◽  
Semen Parameters ◽  
Computer Assisted ◽  
High Concentration ◽  
Bull Semen ◽  
Motile Cells ◽  
Timed Artificial Insemination ◽  
Motility Parameters

The latest entry in the field of semen evaluation is computer assisted semen analysis (CASA). Heterospermic insemination has been used to increase pregnancy rates from low fertile bulls. The objective of this study was to evaluate, with the aid of CASA, heterospermic semen characteristics and pregnancy rates using different concentrations of bull semen in a timed artificial insemination protocol. Semen was collected from two bulls of known fertility by artificial vagina and all CASA motility parameters were evaluated individually and combined. Straws were filled using a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) as follows: single bull A and single bull B (12 × 106 of progressive motile cells after thawing); Mixed bull semen: A + B (12 × 106 of progressive motile cells after thawing) and Supermix bull semen: A + B (28 × 106 of progressive motile cells after thawing). All cows received a P4 intravaginal device (DIB, Syntex, Argentina) and 2 mg of EB i.m. (Syntex) on Day 0, 500 mg cloprostenol (Estroplan, Syntex) at the time of DIB removal (Day 8), and 1 mg EB i.m. on Day 9. Fixed-time insemination (FTAI) was performed at 52 to 56 h after DIB removal. A total of 249 cows were randomly allocated to be inseminated with bulls A and B (n = 76), with Mixed A + B (n = 87), and with Supermixed A+B at a high concentration (n = 86) by a single inseminator. Pregnancy rates were evaluated at 38 days after insemination by transrectal ultrasonography. Means and standard deviations or each characteristic were calculated, compared, and statistically analyzed. The following sperm motility parameters were determined with the Ceros 12.1 sperm analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) on at least 1000 spermatozoa: velocity average path (VAP), velocity straight line (VSL), curvilinear velocity (VCL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of rapid or static cells. There were no significant differences (P > 0.05) in VAP, VSL, VCL, ALH, STR, or LIN. There was a numerically higher percentage of rapid cells in the Supermix semen. Pregnancy rate from bulls A and B was 61% and from Mixed A + B 60%, while that from Supermixed A + B was 69%. Results from the analysis indicate that semen concentration is an important element to be considered in a timed artificial insemination program. Numerically higher pregnancy rates were obtained with double semen concentration in the straw. More research is required to evaluate the interaction between different breeds within a timed artificial insemination program. This research was supported by Centro Genético Bovino de EOLIA sa and Syntex sa Argentina.

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