130 EFFECTIVENESS OF BUTYLATED HYDROXYTOLUENE AS EGG YOLK SUBSTITUTE FOR CRYOPRESERVATION OF BOAR SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 182 ◽  
Author(s):  
L. Rodriguez-Vilar ◽  
M. Hernandez ◽  
C. Lopez-Sanchez ◽  
J. M. Vazquez ◽  
E. A. Martinez ◽  
...  

Butylated hydroxytoluene (BHT) has proven to be efficient as a supplement for cryopreservation boar spermatozoa (Roca et al. 2004 J. Androl. 25, 397–405). Moreover, it has been successfully used as an egg yolk substitute to cryopreserve goat spermatozoa (Khalifa and El-Saidy 2006 Anim. Reprod. Sci. 93, 303–315). The objective of this study was to evaluate the effectiveness of BHT as an egg yolk substitute for freezing boar spermatozoa. Nine sperm-rich ejaculate fractions were collected from 3 boars (3 ejaculates per boar) using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet of each ejaculate was split into 5 aliquots. The aliquots were diluted (to a final concentration of 1 � 109 sperm/mL) in a Tris-citric-glucose extender with 3% glycerol and supplemented with 20% egg yolk (positive control, PC aliquot) or BHT at the final concentrations of 0 (negative control, NC aliquot), 0.2, 0.4, and 0.8 mM. Diluted semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20�C min. Thawing was carried out in a water bath at 70�C for 8 s. Post-thaw sperm survival was assessed according to total sperm motility (TSM, %) using a CASA system (SCA�; Microptic, Barcelona, Spain), and plasma membrane integrity (PMI, %) and acrosome membrane integrity (AMI, %) using a flow cytometric procedure (SYBR-14/propidioum iodide/FITC-phycoerythrin), at 30 and 150 min post-thawing in diluted Beltsville thawing solution with spermatozoa held in a waterbath at 37�C (3 straws per ejaculate). Data were analyzed using a ANOVA mixed model including the main effects of aliquot, boar, post-thaw assessment time, and their interactions, with ejaculate and straw as random effects. All main effects had significant influence (P ≤ 0.01) in all post-thaw sperm assessments. However, no interactions (P ≥ 0.05) among main effects were shown. Data were combined for the 2 post-thaw assessment times. The best (P ≤ 0.05) post-thaw sperm quality (mean � SEM) was achieved in PC aliquots (47.11 � 3.10, 58.98 � 2.78, and 51.35 � 3.42 for TSM, PMI, and AMI, respectively). In NC aliquots, the percentage of TSM, PMI, and AMI were always below 1% (P ≤ 0.05). BHT has a beneficial (P ≤ 0.05) effect on post-thaw sperm assessments, and no differences (P ≥ 0.05) among concentrations were shown. The mean post-thaw sperm quality in the BHT aliquots was 8.50 � 0.80, 20.29 � 0.53, and 16.03 � 0.55 for TSM, PMI, and AMI, respectively. On the basis of these data, we can conclude that BHT has a protective effect for boar spermatozoa during the cryopreservation process. However, BHT alone is insufficient to replace the protective effect of egg yolk. This work was supported by CICYT (AGF2005-00706), Madrid, Spain.

2016 ◽  
Vol 12 (2) ◽  
pp. 21-28
Author(s):  
Monika Trzcińska ◽  
Magdalena Baryła

The objective of the study was to determine the effect of butylated hydroxytoluene (BHT) on the quality and fertilizing capacity of frozen-thawed (FT) boar semen. Semen from five boars (36 ejaculates) was resuspended in lactose-egg yolk-glycerol extender supplemented with 0 (control), 1.0 (R1), 1.5 (R2) or 2.0 mM BHT (R3). Sperm quality was assessed based on motility (CASA; TM: total motility; PM: progressive motility), phosphatidylserine (PS) translocation across the plasma membrane (Annexin-V-FLuos Staining Kit) and DNA fragmentation (TUNEL Assay). The FT semen was also used for intrauterine artificial insemination (AI) of synchronized gilts. The fertilizing capacity of the FT semen was assessed on the basis of the gilt insemination rate and the number of morphologically normal embryos. The quality of the preimplantation embryos was determined by observing a TUNEL-positive reaction. The highest percentage of progressive motile and viable spermatozoa was noted in extender R3 (74.8 ±4.4% and 63.7 ±5.8%), as compared with the control (38.3 ±2.8% and 36.1 ±2.6%). The addition of BHT to the extender did not increase early apoptotic changes in the frozen-thawed spermatozoa with respect to the control. Irrespective of the variant of the extender, cryopreservation and thawing did not induce fragmentation in the boar spermatozoa. The highest number of morphologically normal embryos from inseminated gilts was observed in the case of semen cryopreserved in extender supplemented with 1.5 mM BHT. No significant differences were observed in DNA fragmentation in the expanded blastocysts from gilts inseminated with FT semen cryopreserved in the extenders analysed.


2020 ◽  
Vol 32 (2) ◽  
pp. 206
Author(s):  
L. Gavin-Plagne ◽  
L. Boyer ◽  
A. Baudot ◽  
M. Guedes Teixeira ◽  
G. Louis ◽  
...  

Animal-derived products are widely used in sperm cryopreservation for their cryoprotective properties. These components, however, must be replaced because of sanitary risks. STEMALPHA.CRYO3 (Ref. 5617, Stem Alpha), called CRYO3, is a chemically defined preservation medium currently used for freezing human tissue and adult stem cells. The aim of this study was to evaluate the effects of a CRYO3-based medium and of two cooling rates on invitro parameters and invivo fertility of ram sperm. Six rams (Blanche du Massif Central) were subjected to sperm collection four times using an artificial vagina. Sperm were split and frozen in three media: an egg yolk and milk-based medium (positive control), a CRYO3-based medium (tested medium), and a medium without additives (negative control). The two cooling rates were related to the distance between the straws and the surface of liquid nitrogen during the freezing process (5 and 20cm). Sperm membrane integrity (propidium iodide/SYBR-14), acrosome integrity (fluorescein isothiocyanate-peanut agglutinin/propidium iodide; FITC-PNA/PI), and mitochondrial membrane potential (JC-1) were assessed using flow cytometry, whereas functional membrane integrity was assessed using a hypo-osmotic swelling test and motion characteristics were evaluated using computer-assisted sperm analysis. Pregnancy rate, parturition rate, and prolificacy were evaluated after performing laparoscopic inseminations (n=75 ewes). Moreover, we characterised the freezing media thermodynamically using a differential scanning calorimeter. Statistical analyses were performed using R software. Invitro parameters were assessed using a mixed model including the time and the medium as fixed effects and the ram as a random effect. Pregnancy and parturition rates, following a binomial distribution, and prolificacy, assumed to follow a Poisson distribution, were analysed using generalised linear models, including the medium as a fixed effect and the ram as a random effect. Differences with P<0.05 were considered statistically significant. The cooling rates had no significant effect except on the wobble motion parameter. The positive control medium showed significantly higher results than the CRYO3-based medium and the negative control medium for all invitro parameters except for straightness motion parameter. Conversely, field trials showed no significant difference between the media for pregnancy rate (71, 64, and 74%), parturition rate (68, 61, and 74%) and prolificacy (2.0, 2.1, and 1.7), for the positive control, CRYO3-based medium, and the negative control, respectively. This study showed that the product, CRYO3, cannot replace egg yolk and milk in freezing extenders. Moreover, we showed that laparoscopic inseminations allowed a 74% parturition rate due to an easy and inexpensive medium comprising only a Tris buffer and glycerol. Although it could not be used on a large scale, this medium remains an option for international transport or long-term storage of genetic diversity.


2022 ◽  
Vol 43 (2) ◽  
pp. 841-854
Author(s):  
Lucas Emanuel Ferreira Canuto ◽  
◽  
Lorenzo Garrido Teixeira Martini Segabinazzi ◽  
Endrigo Adonis Braga de Araújo ◽  
Luis Fernando Mercês Chaves Silva ◽  
...  

Cooling and freezing processes cause physical and chemical damage to sperm by cold shock and oxidative stress. This study aimed to evaluate the effect of two antioxidants on sperm parameters of cooled and frozen-thawed ram semen diluted in an egg yolk-based extender. Semen was collected from 30 rams and processed in two consecutive experiments to test the inclusion of different concentrations of quercetin and butylated hydroxytoluene (BHT) in an egg yolk-based semen extender. Dimethyl sulfoxide (DMSO) was added as a solvent to the semen extender in a ratio of 1 mL DMSO for 90 mg of quercetin and 1 mL DMSO for 880 mg of BHT. After collection, semen was diluted at 200 × 106 motile sperm/mL (control) and split into different groups in each experiment. In experiment 1, semen was diluted with the extender containing quercetin (Q5, 5 μg/mL; Q10, 10 μg/mL; Q15, 15 μg/mL) or DMSO alone (DMSO1, 0.055 μL DMSO per mL; DMSO2, 0.165 μL DMSO per mL). In experiment 2, semen was diluted with the extender with BHT (BHT1, 0.5 μg/mL; BHT2, 1 μg/mL; BHT3, 1.5 μg/mL) or DMSO alone (DMSO3, 0.375 μL DMSO per mL; DMSO4, 1.125 μL DMSO per mL). After dilution, the semen was divided into two aliquots. Treated ram sperm samples were also subjected to different storage methods. The first set of samples was cooled at 5 °C for 24 h, whereas the second set of samples was frozen-thawed. Sperm motility parameters and plasma membrane integrity (PMI) were evaluated immediately after dilution (0h) and 24 h after cooling and in the frozen-thawed samples via computer-assisted sperm analysis and epifluorescence microscopy, respectively. The inclusion of quercetin or BHT did not affect sperm motility parameters or PMI of fresh, cooled, or frozen-thawed sperm in this study (P < 0.05). However, further studies are needed to test the effects of these antioxidants on the fertility of cryopreserved ram semen.


2007 ◽  
Vol 19 (1) ◽  
pp. 182
Author(s):  
J. Roca ◽  
M. Hernandez ◽  
T. Cremades ◽  
J. M. Vazquez ◽  
E. A. Martinez

One of the most important limiting factors for the efficient commercial application of frozen–thawed semen on pig artificial insemination programs is the significant and consistent inter-boar variability in sperm cryosurvival. The objective of the present study was to evaluate the effectiveness of different cryopreservation conditions (CCs) for freezing and thawing boar spermatozoa, and to determine their suitability for individual boars, with particular reference to those that showed an intrinsic poor sperm cryosurvival. Using a split-ejaculate technique, single ejaculates from 53 boars were suspended in lactose-egg yolk extender containing 2 or 3% final glycerol concentration, packaged in 0.5-mL straws, cooled at rates of 10, 40, or 60�C min-1 using a programmable cell freezer, and stored in liquid nitrogen; the frozen samples were warmed at ≈1200�C min-1 (37�C water bath for 20 s) or ≈1800�C min-1 (70�C for 8 s). The cryopreservation condition including 2% glycerol, 40�C min-1 of cooling, and ≈1200�C min-1 of warming was considered as the control. Frozen–thawed sperm were evaluated at 30 and 150 min post-thawing for sperm motility (CASA system), plasma membrane integrity (SYBR-14 and propidium iodide), and acrosome membrane integrity (FITC-peanut agglutinin and propidium iodide). Data were analyzed using 2 different ANOVA mixed models. Whereas cooling rate had no influence (P ≥ 0.05), glycerol concentration and warming rate, both independently, affected (P ≤ 0.05) all post-thawing sperm assessments. No interaction (P ≥ 0.05) among effects was detected for any of the sperm parameters assessed. Evaluating the combined effect of glycerol concentration and warming rate, the highest post-thaw sperm quality was achieved from the semen samples frozen with 3% of glycerol and thawed at ≈1800�C min-1. Significant differences (P ≤ 0.05) among ejaculates (boars) to support the different CCs were shown in all post-thaw sperm assessments. Three different (P ≤ 0.05) ejaculate (boar) populations, defined by PATN analysis (PATN software package, CSIRO, Canberra, Australia), were identified according to post-thaw sperm assessments in semen samples frozen and thawed using control CC (populations so-called 'good', 'moderate', and 'bad' sperm freezers). Different (P ≤ 0.05) susceptibility in the tolerance of spermatozoa to support the different CCs was found among the ejaculate populations. Whereas spermatozoa from ejaculates considered as 'good' freezers are relatively unaffected (P ≥ 0.05), those from 'moderate' and, mainly, 'bad' freezers are very sensitive (P ≤ 0.05) to the modifications in the CCs. In conclusion, slight modifications in the CCs — glycerol concentration and warming rate for thawing — can improve the sperm cryosurvival of some ejaculates (boars), the improvement being particularly larger in those ejaculates (boars) classified as ' bad' sperm freezers. This work was supported by CICYT (AGF2005-00760), Madrid, Spain.


2017 ◽  
Vol 9 (2) ◽  
pp. 196 ◽  
Author(s):  
Hamed R. Takruri ◽  
Maha S. Shomaf ◽  
Saida F. Shnaigat

This research was conducted to study the protective effect of bee honey on the 7,12-dimethylbenz(a)anthracene (DMBA)- induced breast cancer in rat model. The study consisted of three groups: honey group, positive control group (PC), and negative control group (NC) to which the carcinogen was not administered. All rats were fed the diet recommended by the American Institute of Nutrition for growing rats (AIN-93G), with addition of honey (50 g/kg diet) to the honey group. All Rats were fed their diets ad libitum on 12 hours dark/light cycle. At the age of 50 days all rats in the honey and PC groups were gavaged once by the carcinogen DMBA with a dose of 80 mg/kg body Wt. After three weeks of carcinogen administration, rats were palpated weekly to detect any tumor growth. After 18 weeks, all rats were sacrificed. The palpable structures and the mammary glands along with associated lymph nodes were removed and fixed in saline formalin and prepared for histopathological examination. The results revealed that the honey group diet significantly (p < 0.05) reduced the incidence rate of mammary cancer, palpable tumor multiplicity, tumor size and weight compared to the PC group. In conclusion, multi floral honey has a protective effect against DMBA- induced mammary cancer in the initiation, promotion, and progression stages of DMBA-induced mammary carcinogenesis. However, further research is needed to reveal the mechanisms that might have contributed to the preventive effect of honey against mammary cancer.


Zygote ◽  
2018 ◽  
Vol 26 (4) ◽  
pp. 301-307 ◽  
Author(s):  
José A. B. Bezerra ◽  
Andréia M. Silva ◽  
Patrícia C Sousa ◽  
Lívia B. Campos ◽  
Érica C. G. Praxedes ◽  
...  

SummaryThe aim of this study was to establish a functional freezing–thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen–thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.


2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
F. V. Ramukhithi ◽  
T. R. Netshirovha ◽  
C. Hlungwani ◽  
...  

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.


2019 ◽  
Vol 53 (5) ◽  
pp. 576-583 ◽  
Author(s):  
Daiana Moreli Soares dos Santos ◽  
Juliana Gonçalves Pires ◽  
Aline Braga Silva ◽  
Priscila Maria Aranda Salomão ◽  
Marília Afonso Rabelo Buzalaf ◽  
...  

This study evaluated the effect of titanium tetrafluoride (TiF4) varnish on the development of dentin carious lesions. Bovine root dentin samples were treated for 6 h with: (A) 4% TiF4 varnish (2.45% F); (B) 5.42% sodium fluoride (NaF) varnish (2.45% F); (C) 2% chlorhexidine (CHX) gel – positive control; (D) placebo varnish; or (E) untreated – negative control (n = 4 × biological triplicate, n = 12). Treated dentin samples were exposed to human saliva mixed with McBain saliva (1:50) for the first 8 h in 24-well plates. Thereafter, the medium was removed, and McBain saliva containing 0.2% sucrose was applied for 16 h. From days 2 to 5, McBain saliva with sucrose was replaced daily (37°C, 5% CO2). The demineralization was measured using transverse microradiography, while the effect on biofilm was analyzed using viability, extracellular polysaccharide (EPS), and lactic acid production assays. The data were statistically analyzed (p < 0.05). All treatments (fluorides and CHX) significantly reduced the biofilm viability compared to placebo varnish and negative control. However, none of them was able to reduce the colony-forming unit counting for total microorganism, total streptococci, and Streptococcus mutans. NaF significantly reduced the number of Lactobacillus sp. compared to negative control. No effect was seen on lactic acid production neither on EPS synthesis, except that CHX significantly reduced the amount of insoluble EPS. Both fluorides were able to reduce dentin demineralization compared to placebo varnish and negative control; TiF4 had a better effect in reducing mineral loss and lesion depth than NaF. Therefore, TiF4 varnish has the best protective effect on dentin carious lesion formation using this model.


Author(s):  
P. J. Chaudhary ◽  
A. J. Dhami ◽  
D. V. Chaudhari ◽  
K. K. Hadiya ◽  
J. A. Patel

This study was undertaken on three mature bulls each of Gir cattle and Surti buffalo breeds to evaluate the comparative efficacy of egg yolk based standard TFYG (Tris-citrate-fructose-yolk-glycerol) extender and egg yolk free soybean based commercial extenders Optixcell® (IMV, France) and Andromed® (Minitube, Germany) under split-sample technique. The ejaculates (9/bull) were extended @ 100×106 sperm ml-1 with three extenders and frozen using biofreezer following 4 hr of equilibration. The pooled means of progressively motile sperm observed (irrespective of extenders) at initial, pre-freeze and post-thaw stage in Gir bulls semen were 76.53±0.53, 71.11±0.53 and 39.86±0.90% and in Surti buffalo 80.76±0.39, 74.65±0.45 and 40.35±1.07%, respectively. The corresponding values for live sperm were 75.64±0.76, 69.01±0.97 and 47.99±1.11 % for Gir and 80.90±0.45, 75.76±0.48 and 52.33±0.86 % for Surti buffalo; and those of intact acrosome 94.29±0.25, 90.29±0.27 and 79.29±0.33 % for Gir bulls, and 93.94±0.21, 89.94±0.23 and 78.95±0.26 % for Surti buffalo semen, respectively. The HOS reactive sperm at initial, pre-freeze and post-thaw stage were 76.18±0.74, 71.04±0.76 and 27.90±0.70 % for Gir, and 81.83±0.35, 76.47±0.39 and 27.83±0.68 % for Surti bulls, respectively. The overall mean post-thaw incubation (37°C) survival of spermatozoa observed at 60, 120 and 180 min were 28.40±0.91, 17.78±0.86 and 9.44±0.72% for Gir bulls semen, and 28.01±0.99, 18.40±1.01 and 10.51±0.93% for Surti buffalo semen, respectively. Optixcell was proved superior, and at par with TFYG, than the Andromed in maintaining greater motility, viability, morphology, acrosomal/plasma membrane integrity including post-thaw sperm longevity of cattle and buffalo spermatozoa with significant differences only in sperm motility and post-thaw longevity. The motile, live and HOST reactive sperm were significantly higher in buffalo semen than cattle at initial and pre-freeze stage, but not at post-thaw stage. The results showed that egg yolk free commercial Optixcell extender and egg yolk based TFYG extender were at par in terms of most of the sperm quality traits, hence any one of them can be preferred over Andromed for successful routine cryopreservation of cattle and buffalo semen.


2011 ◽  
Vol 30 (10) ◽  
pp. 1710-1715 ◽  
Author(s):  
Heibatullah Kalantari ◽  
Mohammadtaha Jalali ◽  
Amir Jalali ◽  
Abobakr Salimi ◽  
Foad Alhalvachi ◽  
...  

The efficacy of a crude hydro-alcoholic extract of Cassia fistula (golden shower tree) fruit to protect the kidney against bromobenzene-induced toxicity was studied. Negative control mice received normal saline; positive control mice were given 460 mg/kg of bromobenzene; Cassia fistula treated mice received 200, 400, 600 and 800 mg/kg of Cassia fistula fruit extract followed by 460 mg/kg bromobenzene (daily by oral gavage for 10 days). On the 11th day, the mice were sacrificed, blood samples were obtained to assess blood urea nitrogen (BUN) and creatinine levels, and kidneys were removed for histological examination. We found that bromobenzene induced significant nephrotoxicity reflected by an increase in levels of BUN and creatinine that was dose dependently prevented by the Cassia fistula fruit extract. The nephroprotective effect of the Cassia fistula fruit extract was confirmed by the histological examination of the kidneys. To the best of our knowledge, this is the first study to demonstrate the protective effect of Cassia fistula in nephrotoxicity.


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