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Published: 10 March 2023

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1

Alcaráz, L., M. Hidalgo, M. J. Galvez, D. Acha, I. Ortiz, S. Demyda-Peyrás, C. Gonzales, et al. "Erratum to: 56 SINGLE LAYER CENTRIFUGATION THROUGH PURESPERM® 80 IMPROVES QUALITY OF CRYOPRESERVED DOG SPERMATOZOA." Reproduction, Fertility and Development 25, no. 1 (2013): 175. http://dx.doi.org/10.1071/rdv25n1ab56.

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Density gradient centrifugation with PureSperm® (PureSperm® 40 + PureSperm® 80; Nidacon International, Mölndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm® on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm® 80 can improve the post-thaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400 × 106 sperm mL–1 with CaniPROTM Freeze A plus 20% egg yolk at 22°C. Extended semen was cooled to 5°C within an hour and then diluted to a final concentration of 150–200 × 106 sperm mL–1 in CaniPROTM Freeze B plus 20% egg yolk at 5°C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4 cm above the surface of liquid nitrogen vapors for 10 min, after which they were directly placed in liquid nitrogen. After 24 to 48 h of storage, straws were thawed in a water bath at 37°C for 30 s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm® 80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as mean ± SEM. Significant (P < 0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.65 ± 0.05 v. 83.79 ± 0.13; percentage of progressive motile spermatozoa: 79.38 ± 6.66 v. 54.61 ± 16.11), morphology (86.45 ± 0.01 v. 83.51 ± 0.01), and viability (percentage of viable sperm with an intact acrosome: 58.32 ± 0.04 v. 36.50 ± 0.17; percentage of viable sperm with an acrosome reaction: 2.81 ± 0.01 v. 9.74 ± 0.21). Based on our results, we can conclude that SLC with PureSperm® 80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.

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2

Alcaráz, L., M. Hidalgo, M. J. Galvez, D. Acha, I. Ortiz, S. Demyda-Peyrás, C. Gonzales, et al. "Erratum to: 56 SINGLE LAYER CENTRIFUGATION THROUGH PURESPERM® 80 IMPROVES QUALITY OF CRYOPRESERVED DOG SPERMATOZOA." Reproduction, Fertility and Development 25, no. 3 (2013): 587. http://dx.doi.org/10.1071/rdv25n1ab56_er.

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Density gradient centrifugation with PureSperm® (PureSperm® 40+PureSperm® 80; Nidacon International, Mölndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm® on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm® 80 can improve the post-thaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400×106spermmL–1 with CaniPROTM Freeze A plus 20% egg yolk at 22°C. Extended semen was cooled to 5°C within an hour and then diluted to a final concentration of 150–200×106spermmL–1 in CaniPROTM Freeze B plus 20% egg yolk at 5°C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4cm above the surface of liquid nitrogen vapors for 10min, after which they were directly placed in liquid nitrogen. After 24 to 48h of storage, straws were thawed in a water bath at 37°C for 30s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm® 80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as mean ± SEM. Significant (P<0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.65±0.05 v. 83.79±0.13; percentage of progressive motile spermatozoa: 79.38±6.66 v. 54.61±16.11), morphology (86.45±0.01 v. 83.51±0.01), and viability (percentage of viable sperm with an intact acrosome: 58.32±0.04 v. 36.50±0.17; percentage of viable sperm with an acrosome reaction: 2.81±0.01 v. 9.74±0.21). Based on our results, we can conclude that SLC with PureSperm® 80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.

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3

Kossakowski, J., L. Morrison, and D. Mortimer. "O-179. Evaluation of PureSperm gradients instead of Percoll for human spermatozoa." Human Reproduction 12, Suppl_2 (June 1997): 88–89. http://dx.doi.org/10.1093/humrep/12.suppl_2.88.

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4

Blevins, B. A., M. de la Rey, and N. M. Loskutoff. "7 EFFECT OF DENSITY GRADIENT CENTRIFUGATION WITH TRYPSIN ON THE FERTILIZING CAPABILITY OF BOVINE SPERM." Reproduction, Fertility and Development 20, no. 1 (2008): 84. http://dx.doi.org/10.1071/rdv20n1ab7.

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The goal of this research was to investigate the effect of a novel density gradient centrifugation (DGC) treatment on the fertilizing capability of bovine sperm as compared to a standard method. Domestic bull (Bos taurus) semen was used for AI and the production of embryos from in vivo-matured bovine oocytes. In 2004, a preliminary study compared the novel semen treatment using a trypsinized PVP-coated silica particle suspension (Percoll; Sigma, St. Louis, MO, USA) to a standard method (4� dilution with an egg yolk diluent) on the fertilizing capacity in vivo of bovine sperm (de la Rey et al. 2005 J. Reprod. Fertil. 17, 242 abst). Although not statistically significant (P = 0.69), there were more transferable quality embryos recovered from cows inseminated using the treated sperm method v. control (58.9 v. 43%, respectively). In this report we provide the results of two additional trials utilizing the novel semen treatment and substituting Percoll with a silane-coated silica particle medium containing a recombinant trypsin (r-protease). In the second trial (2005), semen samples collected from three bulls were processed by DGC: 2 mL of 40% PureSperm (NidaCon International AB, M�lndal, Sweden) containing recombinant trypsin (TrypLE Select, Gibco/Invitrogen, Carlsbad, CA, USA), which overlaid 2 mL of 80% PureSperm containing 10 µg mL–1 soy-based protease inhibitor (Sigma), was overlaid with the semen sample using a novel centrifuge tube insert (ProInsert, Nidacon) and then centrifuged at 300g for 20 min. The sperm pellets were recovered and washed (500g for 10 min) in 10 mL pre-warmed TL-HEPES medium (Cambrex Corp., East Rutherford, NJ, USA). The washed sperm pellets were then resuspended in the same total volume of pre-warmed Biladyl� A (Minit�b, Tiefenbach, Germany) as the standard method and used to AI a total of 42 (control) and 47 (treatment) superovulated cows three times at 12 h intervals. Day 7 embryos were recovered and assessed for stage and morphological quality. In Trial 3 (2006), semen samples collected from three bulls were processed by DGC containing r-protease and a soybean protease inhibitor (BoviPure Pro, NidaCon), media specifically formulated for domestic bull semen. Sperm pellets were washed in 10 mL BoviWash medium (NidaCon). The washed sperm pellet was resuspended in the same total volume of pre-warmed Biladyl A as the standard method and used to AI a total of 23 (control) and 25 (treatment) superovulated cows and embryos evaluated as in Trial 2. The results between control and treated groups were compared using the Mann-Whitney (Wixcoxon rank sum) test. Trial 2 using PureSperm tended to result in higher fertilization rates than for cows inseminated using the standard method (75.2% v. 67%, respectively) but the results were not statistically significant (P = 0.63). Results for Trial 3 indicated that cows inseminated with BoviPure Pro-treated sperm had significantly increased fertilization rates as compared to the standard method (88.4% v. 63.1%, respectively; P = 0.02) and had higher numbers of transferable quality embryos (70.3% v. 51.8%, respectively; P = 0.38). In summary, BoviPure Pro sperm treatment before AI significantly increases fertilization rates and can result in as much as an 18.5% increase in transferable quality embryos as compared to standard methods.

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5

Dorado, J., L. Alcaráz, N. Duarte, J. M. Portero, D. Acha, S. Demyda, A. Muñoz-Serrano, and M. Hidalgo. "Centrifugation on PureSperm® density-gradient improved quality of spermatozoa from frozen-thawed dog semen." Theriogenology 76, no. 2 (July 2011): 381–85. http://dx.doi.org/10.1016/j.theriogenology.2011.02.026.

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6

Perals, M. L., M. A. Gil, E. M. Garcia, J. Sanchez-Osorio, J. M. Vázquez, E. A. Martinez, and J. Roca. "88 SELECTION OF A CRYOSURVIVAL SPERM POPULATION DOES NOT IMPROVE THE IN VITRO PENETRATING ABILITY OF FROZEN - THAWED BOAR SPERMATOZOA." Reproduction, Fertility and Development 20, no. 1 (2008): 124. http://dx.doi.org/10.1071/rdv20n1ab88.

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Boars can be classified as good or bad sperm freezers according to their sperm cryosurvival. Different sperm selection techniques, such as PureSperm� (PS; MidAtlantic Diagnostics, Inc., Mount Laurel, NJ, USA), have been developed to improve functional competence of spermatozoa. The aim of this experimental study was to assess the ability of PS for improving the in vitro penetrating ability of frozen–thawed boar spermatozoa from good and bad sperm freezers. The sperm-rich fractions from two boars, good (Boar A) and bad (Boar B) freezers, were extended in a lactose/eggyolk/ glycerol/Equex Stem (Noba Chemical Sales, Inc., Scituate, ME, USA) mixture (1 � 109 sperm mL–1), dispensed into 0.5-mL straws, and frozen using a programmable cell freezer. After thawing (1.200�C min–1), semen from each boar was split into two aliquots of 500 µL. One aliquot was used as the control. The second was placed into a tube of PS gradient (90%/45%) and centrifuged at 425g for 20 min; the pellet re-suspended in 1 mL of BTS and re-centrifuged at 320g for 10 min (PS sample). Control and PS samples were diluted in supplemented TCM-199 (TCMm; Roca et al. 1998 Reprod. Fertil. Dev. 10, 479–485) at 200 � 106 sperm mL–1. Sperm survival (SV) was assessed afterTCMm dilution according to progressive sperm motility (PSM, %) using a computer-assisted sperm analysis (CASA) system (ISAS�), and plasma and acrosome membrane integrity (PMI; %) by flow cytometry (SYBR�-14/PE-PNA/PI; Molecular Probes, Leiden, The Netherlands). A homologous in vitro penetration (hIVP) assay, using immature oocytes (20 oocytes/2 mL TCMm supplemented with caffeine and calcium lactate), was used to assess sperm penetrating ability (Martinez et al. 1993 Theriogenology 40, 547–557). A total of 960 immature oocytes were inseminated (200 � 103 sperm/oocyte) in 3 batches. After 18 h of co-incubation at 39�C under 5% CO2 in air, the oocytes were washed, mounted on slides, fixed with ethanol:acetic acid (3:1, v/v) for 48 h, stained with 1% lacmoid, and examined under a phase contrast microscope (�400). Oocytes with swollen or unswollen heads of sperm found in the vitellus were considered as penetrated. Sperm penetrability ability (SPA) was assessed according to penetration rate (PR) and the mean number of sperm per oocyte (S/O). Data were analyzed using a PROMIXED model and expressed as mean � SEM. Boar A showed better (P ≤ 0.01) results for both SV and SPA parameters than boar B, independent of sperm treatment. PureSperm improved (P ≤ 0.05) PSM and PMI in both boar A (control v. PS: 48.0 � 5.8 v. 66.5 � 3.6 and 63.1 � 7.7 v. 88.4 � 1.3, respectively) and boar B (12.3 � 1.2 v. 22.2 � 3.7 and 44.3 � 3.5 v. 58.7 � 7.0, respectively). However, no differences (P ≥ 0.05) were observed in PR and S/O in either boar A (71.2 � 3.4 v. 78.3 � 3.1 and 5.0 � 0.4 v. 5.2 � 0.4, respectively) or boar B (34.3 � 3.6 v. 37.3 � 3.9 and 1.5 � 0.1 v. 1.5 � 0.1, respectively). In conclusion, under our laboratory conditions, PureSperm selection improves sperm quality but not in vitro penetrating ability of frozen–thawed spermatozoa of both good and bad sperm freezers. This work was supported by CICYT (AGF2005-00760), Madrid, Spain.

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7

Dorado, J., L. Alcaraz, M. J. Gálvez, D. Acha, I. Ortiz, M. Urbano, and M. Hidalgo. "Single-layer centrifugation through PureSperm® 80 selects improved quality spermatozoa from frozen-thawed dog semen." Animal Reproduction Science 140, no. 3-4 (August 2013): 232–40. http://dx.doi.org/10.1016/j.anireprosci.2013.06.012.

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8

Laursen, Steen B., Claus Yding Andersen, Johnny Hindkjaer, and Karin Erb. "A clinical study comparing PureSperm and SpermFilter for density gradient separation of human spermatozoa in assisted reproduction." Acta Obstetricia et Gynecologica Scandinavica 82, no. 10 (October 2003): 929–35. http://dx.doi.org/10.1034/j.1600-0412.2003.00297.x.

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9

Maxwell, WMC, I. Parrilla, I. Caballero, E. Garcia, J. Roca, EA Martinez, JM Vazquez, and D. Rath. "Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation." Reproduction in Domestic Animals 42, no. 5 (May 14, 2007): 489–94. http://dx.doi.org/10.1111/j.1439-0531.2006.00811.x.

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10

Álvarez-Rodríguez, M., M. Álvarez, L. Anel-López, E. López-Urueña, P. Manrique, S. Borragán, J. M. Morrell, P. de Paz, and L. Anel. "Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm." Theriogenology 85, no. 6 (April 2016): 1097–105. http://dx.doi.org/10.1016/j.theriogenology.2015.11.021.

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11

Setareh badi, Rohoallah, Akram Vatannejad, Heidar Tavilani, Amir Fattahi, and Marziyeh Ghorbani. "Levels of cholesterol, phospholipid and triacyglycerol in subsets of human infertile men spermatozoa isolated by discontinuous PureSperm gradient." Clinical Biochemistry 44, no. 13 (September 2011): S145—S146. http://dx.doi.org/10.1016/j.clinbiochem.2011.08.356.

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12

Alvarez-Rodríguez, Manuel, Mercedes Alvarez, Elena López-Urueña, Carmen Martínez-Rodriguez, Santiago Borragan, Luis Anel-López, Paulino de Paz, and Luis Anel. "Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm® gradient between freeze–thaw cycles." Cryobiology 67, no. 3 (December 2013): 339–46. http://dx.doi.org/10.1016/j.cryobiol.2013.10.001.

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13

Dorado, J., L. Alcaráz, N. Duarte, J. M. Portero, D. Acha, and M. Hidalgo. "Changes in the structures of motile sperm subpopulations in dog spermatozoa after both cryopreservation and centrifugation on PureSperm® gradient." Animal Reproduction Science 125, no. 1-4 (May 2011): 211–18. http://dx.doi.org/10.1016/j.anireprosci.2011.03.013.

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14

Saragusty, Joseph, María Teresa Prieto, Alexandre Courtiol, Romain Potier, Frank Göritz, Thomas B. Hildebrandt, and Robert Hermes. "Sperm rescue in wild African elephants." Reproduction, Fertility and Development 28, no. 9 (2016): 1433. http://dx.doi.org/10.1071/rd14378.

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This study investigated ways of improving the usefulness of ~1700 mL of poor-quality frozen semen collected from wild African elephant (Loxodonta africana) bulls. Ten semen samples from six bulls, frozen with 5% glycerol in Berliner cryomedium, with or without prior removal of the seminal plasma by centrifugation, were tested. All samples were subjected to the following density-gradient centrifugation treatments: no centrifugation (control), sham centrifugation, Percoll, OptiPrep, Isolate and PureSperm. Sample evaluation included motility, concentration, viability, acrosome integrity and normal morphology after thawing and after gradient centrifugation. Motility was also evaluated 3 h after thawing. While all treatments were similar to the Control in acrosome integrity and normal morphology, significant differences were noted in concentration, viability and motility. Samples treated by Percoll showed the best motility, which was maintained unchanged over 3 h of incubation (37°C). Correlations between manual and automated evaluations of concentration were high (cytometer; rho = 0.92), but were lower for viability (cytometer; rho = 0.57) and motility (computer-aided sperm analysis; rho = 0.66). By performing density centrifugation, the quality of these sperm samples may be improved to a level suitable for artificial insemination in elephants. Although a sizeable proportion of cells are lost in the process, combining samples may still allow for multiple inseminations.

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15

Ranganathan, P., and A. Agarwal. "Recovery and survival of sperm is higher with Puresperm density gradient than swim-up in neat and cryopreserved-thawed semen specimens." Fertility and Sterility 76, no. 3 (September 2001): S214. http://dx.doi.org/10.1016/s0015-0282(01)02639-5.

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16

Wu, G. J., and D. C. Ding. "P3.10.16 The comparison of nitric oxide and motion characteristics of two preparation methods in human sperm: 3-layer percoll and puresperm method." International Journal of Gynecology & Obstetrics 70 (2000): C107—C108. http://dx.doi.org/10.1016/s0020-7292(00)85429-6.

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17

Dorado, J., L. Alcaraz, M. J. Gálvez, D. Acha, I. Ortiz, M. Urbano, and M. Hidalgo. "Corrigendum to “Single-layer centrifugation through PureSperm®80 selects improved quality spermatozoa from frozen-thawed dog semen” [Anim. Reprod. Sci. 140 (2013) 232–240]." Animal Reproduction Science 142, no. 3-4 (November 2013): 198. http://dx.doi.org/10.1016/j.anireprosci.2013.09.019.

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18

Amor, Houda, Shelko Nyaz, and Mohamad Eid Hammadeh. "Paternal Smoking in Relation to Sperm Quality and intracytoplasmic Sperm Injection Outcomes." International Journal of Women's Health and Reproduction Sciences 7, no. 4 (January 16, 2019): 451–60. http://dx.doi.org/10.15296/ijwhr.2019.75.

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Objectives: The present study focused on tobacco smoke and its effect on semen parameters, sperm DNA quality (compaction and fragmentation) and clinical outcomes after intracytoplasmic sperm injection (ICSI) therapy Materials and Methods: The semen samples were divided according to smoking status into the following 2 groups, 98 heavy-smokers (G1) and 43 non-smokers (G2). Semen was prepared and purified using the PureSperm gradients according to the WHO guidelines 2010. Protamine deficiency (CMA3 positivity) was assessed by chromomycin CMA3 staining and sperm DNA fragmentation (sDF) by TUNEL assay. Results: The mean concentration and the total motility were significantly higher in G2 in comparison to G1 (P=0.014, and P=0.026 respectively) and the results were similar for the mean percent of the progressive motility and normal morphology (P=0.0001). CMA3+ and sDF in G2 were significantly lower in comparison to G1 (20.35 ± 13.34% vs. 33.30 ± 22.33%, P=0.001; 14.23 ± 13.07% vs. 26.68 ± 19.77%, P=0.0001). Meanwhile, there were no significant differences in the ICSI outcomes, except for the pregnancy rate, which was significantly higher in G2 than in G1 (0.60 ± 0.49% vs. 0.38 ± 0.48%; P=0.013). In G1, CMA3+ correlated negatively with sperm concentration (r=-0.233, P=0.021) but positively with sDF (r=0.484, P=0.0001). In G2, sDF correlated negatively with progressive motility and morphologically normal spermatozoa (r=-0.304, p=0.047; r=-0.361, P=0.017 respectively). Conclusions: The findings of this study revealed that tobacco smoking altered sperm parameters and later affected the pregnancy results in ICSI therapy. CMA3 and TUNEL tests are therefore useful as a supplementary test before any ART treatment to ensure a good prognosis.

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19

Nicolas, M., M. Alvarez, S. Borragán, F. Martinez-Pastor, C. A. Chamorro, M. Alvarez-Rodriguez, P. de Paz, and L. Anel. "Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa." Theriogenology 77, no. 6 (April 2012): 1119–28. http://dx.doi.org/10.1016/j.theriogenology.2011.10.016.

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20

Lambard, S., I. Galeraud-Denis, PT Saunders, and S. Carreau. "Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors." Journal of Molecular Endocrinology 32, no. 1 (February 1, 2004): 279–89. http://dx.doi.org/10.1677/jme.0.0320279.

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It is now well established that oestrogens play a part in germ cell function. These hormones are synthesised by the cytochrome P450 aromatase (P450 arom) and act via two kinds of receptor (ERalpha and ERbeta). Although the presence of aromatase and oestrogen receptors in mammalian testis is now well documented, the localisation of these proteins in human germ cells is not yet clear. The primary purpose of the current study was to look for the expression of aromatase and oestrogen receptors in human germ cells. Human immature germ cells were collected from semen samples with an excess of rounds cells (>20%) and purified spermatozoa were obtained after sedimentation on a discontinuous PureSperm gradient. Expression of aromatase and oestrogen receptors was determined by RT-PCR with specific primers, and by Western blot using monoclonal antibodies. RT-PCR products for aromatase, ERalpha and ERbeta were amplified from total RNA isolated from human germ cells and spermatozoa. We identified an ERalpha isoform variant that lacks exon 4 in human germ cells and visualised P450 arom as a single band of 49 kDa in germ cells, as we have already reported for human ejaculated spermatozoa. By Western blot, we identified two proteins for ERbeta at approximately 50 and approximately 60 kDa, which could correspond to the long and short forms of ERbeta formed from the use of alternative start sites. In human ejaculated spermatozoa, ERbeta protein was not detected, even though we could amplify mRNA. Using Western blot analysis and a monoclonal antibody specific for ERalpha, we detected two proteins in human immature germ cells: one of the expected size (66 kDa) and a second one of 46 kDa. In mature spermatozoa, only the 46 kDa band was observed and we speculate it may be related to the ERalpha isoform lacking exon I. In conclusion, we have identified P450 arom and ER proteins (full-length and variant) in human germ cells. Further studies are now required to elucidate the mechanism of action of oestrogens on human male germ cells, in terms of both genomic and 'non-genomic' pathways.

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21

Ferré, L., Y. Bogliotti, J. Chitwood, M. Kjelland, and P. Ross. "234 HORMONAL FOLLICLE STIMULATION IN HOLSTEIN COWS FOR IN VITRO EMBRYO PRODUCTION USING SPERM SORTED BY FLOW CYTOMETRY." Reproduction, Fertility and Development 28, no. 2 (2016): 248. http://dx.doi.org/10.1071/rdv28n2ab234.

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Transvaginal ultrasound needle-guided ovum pick-up (OPU) and in vitro embryo production (IVP) offer a reliable alternative to conventional embryo transfer to produce offspring. The success of OPU/IVP greatly depends on the number and quality of retrieved oocytes. The aim of this study was to compare OPU/IVP performance from stimulated Holstein cows. Holstein (Bos taurus) >8-year-old pluriparous open dry cows (n = 28) were used for OPU as oocyte donors. Follicular waves in all groups were synchronized by gonadotropin-releasing hormone (GnRH), prostaglandin F2α (PGF), and CIDR administrated on Day 0, followed by stimulation treatments 48 h later. No pre-synch was used. Total hormone dosage were administrated as follows: Group 1: pFSH = 180 mg (Folltropin, Bioniche, Belleville, ON, Canada; n = 7), Group 2: pFSH/LH = 500 IU (Pluset, Calier, Barcelona, Spain; n = 7), Group 3: eCG = 1500 IU (eCG, Biogénesis-Bagó, Buenos Aires, Argentina; n = 7) and Group 4: Control (n = 7), no stimulation. All injections were performed intramuscularly (i.m.) twice a day, during three days. OPU was performed 48 (Group 1) or 24 h (Group 2 and 3) after the last injection. The control group received saline solution i.m. Follicles were classified according to diameter in 4 categories: small (2–5 mm); medium (6–9 mm); large (10–14 mm) and extra large (>15 mm). A Mindray DP-30 Vet (Mindray Medical, Shenzhen, China) was equipped with a micro-convex transducer 5.0- to 8.5-MHz probe along with a disposable 21G needle. The OPU flow rate was 15 mL min–1. Retrieved oocytes were classified according to IETS guidelines as viable (grade 1 + 2) and non-viable (grade 3 + 4). The IVP protocol was according to that in Reprod. Fertil. Devel. (2004, 16, 253). Fertilization (Day 0) was carried out using female sex-sorted semen selected with a discontinuous density gradient (PureSperm, Nidacon, Mölndal, Sweden) and diluted to 1 × 106 sperm mL–1. ANOVA was used for comparisons of mean values and a chi-squared test was used for proportions. Results are presented in the Table 1. In conclusion, pFSH stimulation before ovum pick-up in Holstein cows increased the number of collected and viable oocytes, cleavage, embryo development, and hatching rates in comparison to other follicle stimulation hormones and non-stimulation. A cost-benefit analysis of these methods could be valuable in order to inform whether or not a stimulation protocol is necessary for a commercial IVP operation. Table 1.Numbers of follicles, collected and viable oocytes, cleavage rate, blastocysts and hatching rate

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22

Licea, M. G., J. E. H. Pichardo, J. L. Rodríguez, A. García-Contreras, B. C. Rosales, M. Palma-Irizarry, S. Romo, and M. E. Kjelland. "130 In Vitro Production of Hybrid Desert Bighorn×Domestic Sheep Embryos Using Frozen–Thawed Epididymal Semen from a Hunter-Harvested Ram." Reproduction, Fertility and Development 30, no. 1 (2018): 205. http://dx.doi.org/10.1071/rdv30n1ab130.

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Although considered a species of least concern by the International Union for Conservation of Nature red list, the Desert Bighorn sheep (Ovis canadensis nelsoni) is listed in Appendix II of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) in Mexico, due to population size and the lack of protected areas. Postmortem epididymal sperm collected from a hunter-harvested Desert Bighorn sheep ram in Mexico, with an unofficial Safari Club International score of 197 2/8 and an estimated 5.5 years old, were used to evaluate the in vitro production (IVP) of embryos using postmortem-collected ram sperm. Testicles with epididymides were placed in the refrigerator ~45 min after harvest. Sperm were extracted from each epididymis and assessed separately for total motility (TM), progressive motility (PM), and membrane integrity using a phase contrast microscope. The sperm suspension was obtained from the distal end of both epididymides and cryopreserved 12 h postmortem using triladyl with egg yolk. Membrane integrity and morphology were evaluated using Eosin-Nigrosin stain. Sperm DNA fragmentation was analysed using the Halomax kit (Halosperm SL, Madrid, Spain) with fluorescence microscopy. Centrifugation with density gradient PureSperm (Nidacon International, Mölndal, Sweden) was used to remove dead sperm and debris before IVF. Ovaries were collected from Domestic sheep (Ovis aries) at a local slaughterhouse. The maturation medium was TCM-199 with Earle’s salts and a modified Tris-buffered medium was used for fertilization. Frozen straws of sperm from the Desert Bighorn ram were thawed for 45 s at 37°C. Sperm were diluted with modified Tween medium B with milk powder (mTBM) to a final concentration of 5 × 106 cells mL−1. The gametes were co-incubated for 18 h under previously described conditions. The cumulus cells were mechanically removed from zygotes and grown using a co-culture with granulosa cells in sequential media SOF1-SOF2. With regard to sperm collection, epididymis 1 produced 29 straws of sperm (0.25 mL, 136 × 106 sperm mL−1) and epididymis 2 produced 32 straws of sperm (0.25 mL, 68 × 106 sperm mL−1). The sperm sample used for IVF had TM of 60% and PM of 30%. Live dead staining of fresh sperm showed 68% live (i.e. intact cell membranes) and 28% post-thaw. Regarding DNA integrity, only 2% of sperm had DNA fragmentation at 0 h. Of 15 Grade 1 oocytes used for IVF, 4 cleaved (27%), with 1 developing to blastocyst stage (25%). The results show that frozen–thawed epididymal sperm collected from a recently deceased Desert Bighorn ram can provide a valuable source of sperm for IVP of embryos. These results also provide new information on Desert Bighorn sheep reproductive parameters for use in health assessment, or reproduction and conservation management through gene banking and assisted reproductive techniques.

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Ferré, L. B., C. Fresno, M. E. Kjelland, and P. J. Ross. "29 Development and survival of bovine vitrified sexed IVF-derived embryos in vitro matured with pituitary or human recombinant follicle-stimulating hormone." Reproduction, Fertility and Development 31, no. 1 (2019): 140. http://dx.doi.org/10.1071/rdv31n1ab29.

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Improving in vitro production efficiency involves the development of effective oocyte in vitro maturation conditions. Although >80% of cumulus-oocyte complexes (COC) undergo nuclear maturation, only approximately 30 to 35% of immature bovine COC develop to the blastocyst stage. Also, animal-sourced FSH is typically used in IVF, so an effective alternative using recombinant DNA technology is desirable. The aim of this study was to compare the effect of porcine (p) and recombinant human (rh)FSH concentrations on in vitro performance and post-thaw survival. Ovaries were collected from an abattoir and oocytes were aspirated from 2- to 6-mm follicles. The COC with compact and complete cumulus cell layers were selected and matured in groups of 25 COC in 200µL of M199 medium supplemented with alanyl-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µg mL−1), epidermal growth factor (50ng mL−1), pLH (5µg mL−1), cysteamine (0.1mM), and 10% FBS for 22 to 24h in humidified air and 5% CO2. Oocytes were divided into the following groups: 1× pFSH (2µg mL−1), 1× rhFSH (0.01 UI mL−1), 1× pFSH+1× rhFSH, 2× pFSH, and 2× rhFSH. After 22 to 24h, fertilization (Day 0) was carried out using female sexed-sorted semen selected with a mini single-continuous 80% layer (PureSperm, Nidacon International AB, Mölndal, Sweden) and diluted to 1×106 sperm mL−1. The SOF-FERT medium was supplemented with fructose (90µg mL−1), penicillamine (3µg mL−1), hypotaurine (11µg mL−1), and heparin (20µg mL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. Also, 2% FBS was added post-fertilization on Day 3.5. Expanded blastocysts were selected based on IETS standards at Day 6.5 to 7 of culture. Only grade 1 expanded blastocysts were vitrified (Cryotop, Kitazato, Tokyo, Japan). Vitrification medium was 15% (vol/vol) ethylene glycol+propylene glycol. Vitrified embryos were thawed in a solution of H199+20% FBS and 0.25M sucrose at 39°C. Thawed embryos were cultured in SOF-BSA+10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0h), re-expansion and development progress (24-48h), and hatching of the zona pellucida (72h). A minimum of 4 replicates were performed. Data were analysed using a generalized linear mixed model with logit-link binomial distribution. Media treatment differences were determined using Fisher’s least significant difference test with the Bonferroni correction (α-level=0.05). The FSH origin affected cleavage and embryo development rate but not cryotolerance (Table 1). The results support previous research on low dose versus high dose rhFSH effectiveness and interspecies interaction of FSH on follicular receptors. Table 1.Cleavage, embryo development rate, and cryotolerance of FSH of various origins

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Curnow, E. C., and E. S. Hayes. "264IN VITRO FERTILIZATION OF MACACA NEMESTRINA OOCYTES WITH FRESH AND FROZEN-THAWED EPIDIDYMAL SPERM." Reproduction, Fertility and Development 16, no. 2 (2004): 252. http://dx.doi.org/10.1071/rdv16n1ab264.

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In an effort to develop methods to maximize the use of reproductive tissues in our own tissue distribution program (WaNPRC TDP) and to serve as platforms for application of ART to endangered non-human primate (NHP) species, we have examined the effects of collection and cryostorage on epididymal sperm (ES) collected from Macaca nemestrina (Mn). Fresh ES was collected by needle aspiration from the cauda epididymis and prepared by centrifugation in mHTF (Irvine Scientific, Santa Ana, CA, USA)+BSA (3mgmL−1) at 700g for 10min. The resultant pellet was resuspended in fresh mHTF+BSA and held at RT until required. ES subjected to freeze-thaw was collected from the cauda epididymis of testes provided by the WaNPRC TDP by slicing and swim-out into mHTF+BSA. ES was frozen in mTTE medium+5% glycerol (Sankai T et al., 1994 J. Reprod. Fertil. 101, 273). Frozen ES straws were removed from liquid nitrogen and thawed in air at RT for 10min. Thawed ES was centrifuged through an 80% PureSperm gradient (Spectrum Technologies, Healdsburg, CA, USA) at 700g for 15min. The resultant pellet was retrieved and washed in mHTF+BSA at 600g for 5min. For capacitation sperm were incubated in pre-equilibrated HTF+BSA containing 1.0mM caffeine and 0.1mM dbcAMP (Sigma, St. Louis, MO, USA) for 5–10min in a humidified atmosphere of 6% CO2 in air. In vitro-matured Mn oocytes collected from unstimulated ovaries were exposed to fresh or frozen-thawed ES whereas in vivo-matured Mn oocytes collected from superovulated ovaries were exposed to fresh ES in 50-μL Fertilized oocytes were cultured for 24–48h and assessed for cleavage on Day 3 post-insemination. Data were analyzed by single-factor ANOVA or t-test where appropriate and are expressed as mean±SD. Fresh Mn ES (n=4) exhibited very low motility at collection (38±10%) but recovered motility following processing (58±20%). Frozen-thawed ES (n=4) also exhibited low motility post-thaw (53±17%) and recovered after processing (66±28%). The percentage of acrosome-intact sperm was not significantly different (P>0.05) for fresh ES (58.9±8.6%) compared to frozen-thawed ES (70.6±21.9%). Fresh and frozen-thawed ES fertilized in vitro-matured Mn oocytes at similar rates (fresh 68±10.3%, n=170 v. frozen-thawed 71.2±6.7%, n=90; P>0.05). Cleavage rates of fertilized IVM oocytes were not significantly different (fresh 79±7.2% v. frozen-thawed 79.8±5.0%; P>0.05). Fresh ES was also able to fertilize in vivo-matured Mn oocytes collected from superovulated ovaries (95.1±2.5%, n=98) and fertilized oocytes went on to cleave at a high rate (96.1±2.6%). These results suggest that fresh and frozen-thawed ES may be useful for applied ART in endangered species. This work was supported by NIH grant #RR00166 and the WaNPRC TDP.

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Ferré, L., M. Kjelland, T. Stroud, and P. Ross. "107 Follicular wave synchronization and FSH stimulation prior to ovum pickup for invitro embryo production." Reproduction, Fertility and Development 32, no. 2 (2020): 180. http://dx.doi.org/10.1071/rdv32n2ab107.

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Invitro embryo production (IVP) has become a reliable alternative for genetic improvement in beef and dairy herds. Maximizing embryo yield and pregnancy per donor are key factors. The aim of this study was to compare ovum pickup (OPU) yields, developmental competence of cumulus-oocyte complexes (COCs), and pregnancy rates from Angus donors in a commercial IVP setting. Donors (>4-year-old pluriparous open dry cows) were handled under the same feeding and environmental conditions. Treatment groups were organised as follows: Group 1: no synchronization (SYNCH; n=5); Group 2: SYNCH with no superstimulation (SOV; n=5); Group 3: SYNCH + SOV (n=5) and OPU 36h after last FSH injection; Group 4: SYNCH + SOV (n=5) and OPU 48h after last FSH injection; and Group 5: SYNCH + SOV (n=5) and OPU 72h after last FSH injection. Follicular waves in groups 2, 3, 4, and 5 were synched by gonadotrophin-releasing hormone (GnRH), prostaglandin F2α (PGF), and controlled internal drug release (CIDR). No pre-synch was used. Injections of FSH (pFSH=180mg, Folltropin) were performed IM twice a day, for three days. A minimum of three replicates were performed for each donor. A Mindray DP30V equipped with a micro-convex transducer 5.0-8.5MHz probe, disposable 20-gauge needle, and a flow rate of 15mL min−1 were used for OPU. All visible follicles (Foll) were punctured and retrieved into a 50-mL 36°C warmed tube with media (phosphate-buffered saline, bovine serum albumin (BSA), and heparin). Viable oocytes were classified according to IETS guidelines. The COCs were matured in 100µL of M199 medium supplemented with ALA-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µgmL−1), epidermal growth factor (50ngmL−1), oFSH (50ngmL−1), bLH (3μgmL−1), cysteamine (0.1mM), and 10% fetal bovine serum (FBS) for 22 to 24h. Fertilization (Day 0) was carried out using highly fertile sires selected by discontinuous 40%/80% layers (PureSperm) and diluted to a final concentration of 1×106 spermmL−1. Matured oocytes were fertilized in 50µL of modified synthetic oviductal fluid (SOF) media supplemented with fructose (90µgmL−1), penicillamine (3µgmL−1), hypotaurine (11µgmL−1), and heparin (10µgmL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in 50-µL drops of SOF-BSA for 7 days. On Day 3.5, 2% of FBS was added. On Day 7, fresh transferable (grade 1 and 2, IETS standards) blastocysts were implanted into synchronized recipient cows. Around Day 30, ultrasound diagnosis was performed to determined pregnancy rate (PR). We used ANOVA for comparisons of mean values and X2 test for proportions, α=0.05 (Table 1). In conclusion, synchronization, FSH stimulation, and 48-h coasting before OPU in Angus cows increased the number of collected viable oocytes and embryo development rates. More transferrable embryos and higher rates of PR per OPU were obtained using 36- and 48-h coasting, respectively. Table 1.Follicles (Foll), viable oocytes, cleavage, blastocysts, and pregnancy rates (PR) in Angus cows after ovum pickup (OPU) or invitro embryo production (IVP) SYNCH1 FSH Coasting Foll/OPU Oocytes/OPU Viable oocytes Cleavage,% Embryos,% Embryos/OPU PR,% PR/OPU NO NO NO 10.8a 9.0a 8.9a 63a 25a 2.3a 45a 1.0a YES NO NO 10.1a 8.4a 8.1a 73b 27b 2.2a 51b 1.1a YES YES 36H 16.5b 13.8b 13.6b 70c 29b 4.0b 46a 1.8b YES YES 48H 16.1b 13.4b 13.3b 69c 28b 3.8c 52b 2.0b YES YES 72H 15.6b 13.0b 12.6b 64a 22c 2.8d 55b 1.6c a-dValues with different superscripts in the same column differ (P<0.05). 1SYNCH=synchronized.

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Mundodi, Suparna, Porecha Rishi, Anderson Murray, and Rapicavoli Nicole. "FPLC in a pipette tip (P3297)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 211.6. http://dx.doi.org/10.4049/jimmunol.190.supp.211.6.

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Abstract Molecular characterization of the immune response and how this process is implemented requires the purification of native proteins and antibodies. There are numerous purification methods to isolate proteins of interest, but these methods usually require multiple steps to attain the level of purity required for most studies. The Rainin PureSpeed Protein Purification System utilizes pipette tips containing purification resin at their distal end interfaced with an E4 XLS pipette to simplify enrichment procedures for virtually all proteins. The E4 XLS pipette, when set up within a 96-deepwell plate, is able to carry out semi-automated purification of antibodies and other native or recombinant proteins. The pipette is able to drive up-and-down sample flow over the immobilized resin matrix for as many as 12 samples in parallel. The ability of the pipette to carry out liquid handling steps enhances laboratory productivity by allowing an investigator to step away from the apparatus until the next step in the protocol. This new, innovative technology from Rainin Instrument is available with pipette tips containing protein A, protein G, Ni-IMAC, or one of four different ion exchange resins, which allow for multiple options for protein purification. Finally, the PureSpeed System functions well in advanced applications such as protein and chromatin immunoprecipitation, offering speed and versatility relative to agarose and magnetic bead technologies.

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Hernandez-Lopez, L., N. Umland, R. Mondragon-Ceballos, and P. L. Nayudu. "Comparison of the effects of Percoll and PureSpermR on the common marmoset (Callithrix jacchus) semen." Journal of Medical Primatology 34, no. 2 (April 2005): 86–90. http://dx.doi.org/10.1111/j.1600-0684.2005.00095.x.

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Sefrioui, Mohamed Réda, Ibrahim Sbai El Othmani, Halima Filali, Sanae Derfoufi, Soufiane Derraji, Adnane Benmoussa, and Amal Ait Haj Said. "Evaluation of spermicidal activity of saponosides from Saponaria officinalis / Caryophyllaceae, Glycyrrhizia glabra / Fabaceae and Herniaria glabra / Caryophyllaceae." Medicine and Pharmacy Reports, February 15, 2021. http://dx.doi.org/10.15386/mpr-1879.

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Background and objective. Chemical spermicides currently marketed and widely used are known to have many side effects. Thereby, and in order to look for more tolerated natural spermicidal agents, the aim of this work was to evaluate the spermicidal potential of saponin extracts from the roots of Saponaria officinalis / Caryophyllaceae, Glycyrrhizia glabra / Fabaceae, and Herniaria glabra / Caryophyllaceae by studying their in vitro effects on sperm mobility and vitality. Methods. Methanolic saponin extracts from the plants roots were performed. Sperm suspensions were prepared by centrifugation on a PureSperm® density gradient (70 and 45%) and incubated with various concentrations of saponin extracts (50, 250, 500 and 750 mg/mL) at 37°C. The spermicidal activity was evaluated by studying the mobility and vitality of spermatozoa at different time intervals ranging from 10 to 240 minutes. Results. A dose and time dependent effect on sperm mobility and vitality was observed for our extracts. Extracts from Saponaria officinalis roots induced an irreversible immobilization and a total non-viability of sperm within 10 minutes at a concentration of 750 mg/mL. A similar effect was observed within 30 minutes at 750 mg/mL for Herniaria glabra extract and within 90 minutes at 500 mg/ml for Glycyrrhizia glabra extract. Conclusion. The results of our study showed that the saponin extracts of our plants roots possess potent in vitro dose and time dependant spermicidal effect. These natural products could therefore represent a safer and better tolerated alternative to chemical spermicides.

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