111 Fertilizing ability of frozen and freeze-dried semen following intracytoplasmic sperm injection of invitro-matured sheep oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 182
Author(s):  
I. Menéndez-Blanco ◽  
F. Ariu ◽  
A. Piras ◽  
S. Nieddu ◽  
M. Paramio ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Freeze Drying ◽  
Polar Body ◽  
Internal Diameter ◽  
Sugar Solution ◽  
Fluid Medium ◽  
Male And Female ◽  
Male Pronucleus ◽  
Freeze Dried ◽  
Female Pronucleus

Freeze-drying is a novel technique that permits the storage of semen at room temperature for long time periods, retaining their fertilizing capacity. The main objective of this work was to compare the fertilization ability of frozen-thawed (FT) and freeze-dried (FD) ram semen following intracytoplasmic sperm injection (ICSI) of invitro-matured (IVM) sheep oocytes. Oocytes were recovered by slicing the ovaries of slaughtered sheep. Selected cumulus-oocyte complexes (COCs) were IVM for 24h in tissue culture medium 199 (TCM-199) supplemented with 10% heat-treated oestrous sheep serum (ESS), 0.36mM pyruvate, FSH (1IUmL−1), and luteinising hormone (LH; 1IUmL−1) under mineral oil in a humidified atmosphere of 5% CO2, at 38.5°C. Semen was collected from fertile adult rams using an artificial vagina and processed for (1) freezing and thawing (Khalifa and Lymberopoulos, 2013 Cell Tissue Bank 14, 687-698; https://doi.org/10.1007/s10561-012-9357-6) or (2) freeze-drying and rehydration according to Arav et al. (2018 J. Assist. Reprod. Genet. 35, 1149-115; https://doi.org/10.1007/s10815-018-1145-1) protocols. For FD protocol, sperm samples were diluted in a sugar solution of trehalose and sorbitol (LyoB) and dehydrated for 24h. Later, the samples were rehydrated in a warming solution and diluted in TCM-199 before ICSI. After maturation, metaphase II (MII) oocytes with a polar body were injected with FT or FD sperm. Briefly, oocytes were transferred into groups of six in an ICSI dish containing 6-µL drops of holding medium (TCM-199 + 5% fetal bovine serum) and 3-µL drops of PVP for the sperm samples. Injection was carried out with an inverted microscope (Olympus IX73) connected to a micromanipulation system (Narishige) using ICSI pipettes with 7-µm internal diameter. Within 1h, ICSI oocytes were activated with 5 µM ionomycin for 4min and invitro cultured in modified synthetic oviductal fluid medium (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817; https://doi.org/10.1071/RD11023). After 17-21h, injected oocytes were fixed and stained in a solution of ethanol Hoechst 33342 and classified as FPN (one female pronucleus and one condensed sperm head), MPN (one male pronucleus and one MII), 2PN (two pronuclei, male and female), 3PN (three or more pronuclei), and NPN (no pronuclei). Data were analysed using analysis of variance (two-way ANOVA) followed by Tukey post hoc test with SAS software, version 9.4. The ICSI-FD group had a higher number of NPN and a lower number of 2PN than did the ICSI-FT group (P<0.05). We think that more technical advances in the FD process as well as the rehydration procedure are necessary to improve the application of FD ovine semen for invitro fertilization by ICSI in sheep, but in any case these results have showed that FD could be a useful tool for the future of invitro embryo production. Table 1.Pronuclear formation at 17-21h post-injection1 Treatment n FPN MPN 2PN 3PN NPN FT 71 9.66±4.12 4.26±1.48 48.13±2.79a 5.97±4.16 31.98±6.75a FD 65 6.16±2.26 1.39±1.39 20.15±4.14b 10.57±6.59 61.73±6.89b a,bValues in the same column with different superscript letters differ significantly (P<0.05). 1Data are presented as mean±s.e.m. FPN=female pronucleus, MPN=one male pronucleus and one metaphase II oocyte, 2PN=two pronuclei, male and female, 3PN=three or more pronuclei, NPN=no pronuclei. Funding was provided by Spanish MINECO Grant AGL2017-85837-R, Spanish MECD pre-doctoral grant FPU15/00773, and Spanish MECD mobility grant EST18/00472 to Irene Menéndez Blanco.

The Drosophila kinesin-like protein KLP3A is required for proper behavior of male and female pronuclei at fertilization

Development ◽  
1997 ◽  
Vol 124 (12) ◽  
pp. 2365-2376 ◽  
Author(s):  
B.C. Williams ◽  
A.F. Dernburg ◽  
J. Puro ◽  
S. Nokkala ◽  
M.L. Goldberg
Keyword(s):  
Drosophila Melanogaster ◽  
Polar Body ◽  
Female Meiosis ◽  
Gene Encoding ◽  
Male And Female ◽  
Male Pronucleus ◽  
Female Pronucleus ◽  
And Migration ◽  
Sperm Aster ◽  
Major Defect

Drosophila melanogaster females homozygous for mutations in the gene encoding the kinesin-like protein KLP3A are sterile (Williams et al., 1995). We have investigated the basis of this sterility. The eggs produced by KLP3A mutant mothers are fertilized by sperm, and female meiosis appears to occur normally. However, the large majority of these embryos arrest their development soon thereafter with a characteristic phenotype. The four nuclei produced by female meiosis associate together in a polar body-like structure, while a bipolar spindle is established around the metaphase-arrested male pronucleus. Thus, the major defect caused by depletion of the KLP3A protein is either in specification of the female pronucleus, or in migration of the male and female pronuclei toward each other. We have also found that the KLP3A protein is located throughout the metaphase spindle during meiosis and the early embryonic mitotic divisions, but later accumulates specifically at the midzone of these same spindles during telophase. The protein is also present on two other microtubule structures: the sperm aster; and the radial, monastral array of microtubules established between the two meiosis II spindles. We discuss these results in light of possible functions of the KLP3A protein in pronuclear specification and migration.

Fertilization and ooplasmic movements in the ascidian egg

Development ◽  
1989 ◽  
Vol 105 (2) ◽  
pp. 237-249 ◽  
Author(s):  
C. Sardet ◽  
J. Speksnijder ◽  
S. Inoue ◽  
L. Jaffe
Keyword(s):  
Polar Body ◽  
Surface Velocity ◽  
Second Phase ◽  
Animal Pole ◽  
Male Pronucleus ◽  
Vegetal Pole ◽  
Female Pronucleus ◽  
Ooplasmic Segregation ◽  
Second Polar Body ◽  
Sperm Aster

Using light microscopy techniques, we have studied the movements that follow fertilization in the denuded egg of the ascidian Phallusia mammillata. In particular, our observations show that, as a result of a series of movements described below, the mitochondria-rich subcortical myoplasm is split in two parts during the second phase of ooplasmic segregation. This offers a potential explanation for the origin of larval muscle cells from both posterior and anterior blastomeres. The first visible event at fertilization is a bulging at the animal pole of the egg, which is immediately followed by a wave of contraction, travelling towards the vegetal pole with a surface velocity of 1.4 microns s-1. This wave accompanies the first phase of ooplasmic segregation of the mitochondria-rich subcortical myoplasm. After this contraction wave has reached the vegetal pole after about 2 min, a transient cytoplasmic lobe remains there until 6 min after fertilization. Several new features of the morphogenetic movements were then observed: between the extrusion of the first and second polar body (at 5 and 24–29 min, respectively), a series of transient animal protrusions form at regular intervals. Each animal protrusion involves a flow of the centrally located cytoplasm in the animal direction. Shortly before the second polar body is extruded, a second transient vegetal lobe (‘the vegetal button’) forms, which, like the first, resembles a protostome polar lobe. Immediately after the second polar body is extruded, three events occur almost simultaneously: first, the sperm aster moves from the vegetal hemisphere to the equator. Second, the bulk of the vegetally located myoplasm moves with the sperm aster towards the future posterior pole, but interestingly about 20% remains behind at the anterior side of the embryo. This second phase of myoplasmic movement shows two distinct subphases: a first, oscillatory subphase with an average velocity of about 6 microns min-1, and a second steady subphase with a velocity of about 26 microns min-1. The myoplasm reaches its final position as the male pronucleus with its surrounding aster moves towards the centre of the egg. Third, the female pronucleus moves towards the centre of the egg to meet with the male pronucleus. Like the myoplasm, the migrations of both the sperm aster and the female pronucleus shows two subphases with distinctly different velocities. Finally, the pronuclear membranes dissolve, a small mitotic spindle is formed with very large asters, and at about 60–65 min after fertilization, the egg cleaves.

Effect of harvest period on the protein content in sea buckthorn leaves

2003 ◽  
Vol 83 (2) ◽  
pp. 409-410 ◽  
Author(s):  
T. S. C. Li ◽  
D. Wardle
Keyword(s):  
Protein Content ◽  
Total Protein ◽  
Freeze Drying ◽  
Conversion Factor ◽  
Growing Season ◽  
Sea Buckthorn ◽  
Leaf Protein ◽  
Total Protein Content ◽  
Male And Female ◽  
Freeze Dried

A study was conducted at Summerland, BC, in 2000 and 2001 to investigate the effect of harvest period on the protein content of sea buckthorn leaves. Leaves of both male and female plants were collected bi-weekly during the growing season and analyzed with a LECO FP-528 nitrogen analyzer after air- and freeze-drying. A conversion factor of kA = 5.7 was applied to obtain the protein content. Total protein content in sea buckthorn leaves showed very small differences between male and female plants and from either freeze-dried or air-dried materials. There were significant fluctuations among harvest periods in both years. Based on the results of this experiment, sea buckthorn leaves should be harvested from late July to early August since leaf protein content peaked around this period of time and started to decline significantly by the middle of August. Key words: Sea buckthorn, Hippophae rhamnoides L., leaf protein

scholarly journals α-amylase activity in freeze-dried and spray-dried honey

2020 ◽  
Author(s):  
Anete Keke ◽  
◽  
Ingmars Cinkmanis ◽  
Keyword(s):  
High Performance ◽  
Freeze Drying ◽  
Negative Impact ◽  
Amylase Activity ◽  
Drying Methods ◽  
Sugar Solution ◽  
Powder Concentration ◽  
Freeze Dried ◽  
Spray Dried

Honey is a naturally supersaturated sugar solution, which tends to crystallize. The crystallization of honey can lead to unwanted fermentation that can have a negative impact to honey quality. The production of honey powder could be an alternative method to prevent honey from fermentation. Honey powder could be used as alternative substitute to liquid honey that would allow to use this product more widely in the food industry. α-amylase activity is one of the most important parameters to evaluate the quality of honey. The aim of this study was to investigate the effect of freeze-drying and spray-drying on honey α-amylase activity. Detection of α-amylase activity was carried out by spectrophotometric method. High-performance liquid chromatography was used to determine the content of hydroxymethylfurfural in the powders. The obtained results showed that both drying methods had a negative impact to the enzyme activity in the samples. The lowest activity of α-amylase (8.3 DN) was measured in the spray-dried honey powder. Concentration of hydroxymethylfurfural (HMF) in the powders did not exceed required concentration 40 mg kg-1.

scholarly journals Morphology of gametes, post-fertilization events and the effect of temperature on the embryonic development of Astyanax altiparanae (Teleostei, Characidae)

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 795-807 ◽  
Author(s):  
Matheus Pereira dos Santos ◽  
George Shigueki Yasui ◽  
Pedro Luiz Porfírio Xavier ◽  
Nadya Soares de Macedo Adamov ◽  
Nivaldo Ferreira do Nascimento ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Polar Body ◽  
Effect Of Temperature ◽  
Male And Female ◽  
Fundamental Information ◽  
Spherical Head ◽  
Typical Morphology ◽  
Female Pronucleus ◽  
Second Polar Body ◽  
First Time

SummaryThe aim of this study was to describe the morphology of gametes, post-fertilization events and subsequent temperature effects on the early developmental stages of the neotropical species Astyanax altiparanae. The sperm of this species presents a typical morphology of teleost sperm with a spherical head (diameter = 1.88 µm), midpiece (diameter = 0.75 µm) and a single flagellum (length = 18.67 µm). The extrusion of the second polar body and fusion of male and female pronucleus were reported for the first time in this species. Additionally, we observed the formation of the fertilization cone, which prevents polyspermic fertilization. Developmental stages at 22°C, 26°C and 30°C gave rise to fertilization rates at 91.12, 91.42 and 93.04% respectively. Hatching occurred at 25 hpf at 22°C, 16 hpf at 26°C and 11 hpf at 30°C and the hatching rates were 61.78%, 62.90% and 59.45%, respectively. At 22°C, the second polar body was extruded at ≈6 mpf and the male and female pronucleus fused at ≈10 mpf. This fundamental information is important for the field and opens up new possibilities in fish biotechnology, including micromanipulation and chromosome-set manipulation.

scholarly journals Failure of pronuclear migration and repeated divisions of polar body nuclei associated with MTOC defects in polo eggs of Drosophila

2000 ◽  
Vol 113 (18) ◽  
pp. 3341-3350 ◽  
Author(s):  
M.G. Riparbelli ◽  
G. Callaini ◽  
D.M. Glover
Keyword(s):  
Polar Body ◽  
Motor Protein ◽  
Meiotic Spindle ◽  
Wild Type ◽  
Female Meiosis ◽  
Male Pronucleus ◽  
Female Pronucleus ◽  
Sperm Aster ◽  
Meiosis I

The meiotic spindle of Drosophila oocytes is acentriolar but develops an unusual central microtubule organising centre (MTOC) at the end of meiosis I. In polo oocytes, this common central pole for the two tandem spindles of meiosis II was poorly organised and in contrast to wild-type failed to maintain its associated Pav-KLP motor protein. Furthermore, the polar body nuclei failed to arrest at metaphase, and the four products of female meiosis all underwent repeated haploid division cycles on anastral spindles. This was linked to a failure to form the astral array of microtubules with which the polar body chromosomes are normally associated. The MTOC associated with the male pronucleus was also defective in polo eggs, and the sperm aster did not grow. Migration of the female pronucleus did not take place and so a gonomeric spindle could not form. We discuss these findings in relation to the known roles of polo like kinases in regulating the behaviour of MTOCs.

Scanning Electron Microscopy-cuticular Lesions on the Roundworm Ascaris Suum

1970 ◽  
Vol 28 ◽  
pp. 114-115
Author(s):  
P. A. Madden ◽  
W. R. Anderson
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Freeze Drying ◽  
Room Temperature ◽  
Secondary Electron ◽  
Distilled Water ◽  
Freeze Dried ◽  
Silver Paint ◽  
To Come ◽  
Scanning Electron

The intestinal roundworm of swine is pinkish in color and about the diameter of a lead pencil. Adult worms, taken from parasitized swine, frequently were observed with macroscopic lesions on their cuticule. Those possessing such lesions were rinsed in distilled water, and cylindrical segments of the affected areas were removed. Some of the segments were fixed in buffered formalin before freeze-drying; others were freeze-dried immediately. Initially, specimens were quenched in liquid freon followed by immersion in liquid nitrogen. They were then placed in ampuoles in a freezer at −45C and sublimated by vacuum until dry. After the specimens appeared dry, the freezer was allowed to come to room temperature slowly while the vacuum was maintained. The dried specimens were attached to metal pegs with conductive silver paint and placed in a vacuum evaporator on a rotating tilting stage. They were then coated by evaporating an alloy of 20% palladium and 80% gold to a thickness of approximately 300 A°. The specimens were examined by secondary electron emmission in a scanning electron microscope.

Revealing gross three-dimensional structure in the SEM

1987 ◽  
Vol 45 ◽  
pp. 656-657
Author(s):  
Sterling P. Newberry
Keyword(s):  
Freeze Drying ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Small Object ◽  
Final Solution ◽  
Dimensional Representation ◽  
X Ray ◽  
Three Dimensional Representation ◽  
Freeze Dried ◽  
Critical Point Drying

The beautiful three dimensional representation of small object surfaces by the SEM leads one to search for ways to open up the sample and look inside. Could this be the answer to a better microscopy for gross biological 3-D structure? We know from X-Ray microscope images that Freeze Drying and Critical Point Drying give promise of adequately preserving gross structure. Can we slice such preparations open for SEM inspection? In general these preparations crush more readily than they slice. Russell and Dagihlian got around the problem by “deembedding” a section before imaging. This some what defeats the advantages of direct dry preparation, thus we are reluctant to accept it as the final solution to our problem. Alternatively, consider fig 1 wherein a freeze dried onion root has a window cut in its surface by a micromanipulator during observation in the SEM.

scholarly journals Effect of Freeze Drying and Simulated Gastrointestinal Digestion on Phenolic Metabolites and Antioxidant Property of the Natal Plum (Carissa macrocarpa)

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1420
Author(s):  
Faith Seke ◽  
Vimbainashe E. Manhivi ◽  
Tinotenda Shoko ◽  
Retha M. Slabbert ◽  
Yasmina Sultanbawa ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Array Detector ◽  
Gastrointestinal Digestion ◽  
Liquid Chromatograph ◽  
Antioxidant Power ◽  
Intestinal Phase ◽  
Glucosidase Activity ◽  
Freeze Dried ◽  
Small Intestinal ◽  
The Impact

Natal plums (Carissa macrocarpa) are a natural source of bioactive compounds, particularly anthocyanins, and can be consumed as a snack. This study characterized the impact of freeze drying and in vitro gastrointestinal digestion on the phenolic profile, antioxidant capacity, and α-glucosidase activity of the Natal plum (Carissa macrocarpa). The phenolic compounds were quantified using high performance liquid chromatography coupled to a diode-array detector HPLC-DAD and an ultra-performance liquid chromatograph (UPLC) with a Waters Acquity photodiode array detector (PDA) coupled to a Synapt G2 quadrupole time-of-flight (QTOF) mass spectrometer. Cyanidin-3-O-β-sambubioside (Cy-3-Sa) and cyanidin-3-O-glucoside (Cy-3-G) were the dominant anthocyanins in the fresh and freeze-dried Natal plum powder. Freeze drying did not affect the concentrations of both cyanidin compounds compared to the fresh fruit. Both cyanidin compounds, ellagic acid, catechin, epicatechin syringic acid, caffeic acid, luteolin, and quercetin O-glycoside from the ingested freeze-dried Natal plum powder was quite stable in the gastric phase compared to the small intestinal phase. Cyanidin-3-O-β-sambubioside from the ingested Natal plum powder showed bioaccessibility of 32.2% compared to cyanidin-3-O-glucoside (16.3%). The degradation of anthocyanins increased the bioaccessibility of gallic acid, protocatechuic acid, coumaric acid, and ferulic acid significantly, in the small intestinal digesta. The ferric reducing antioxidant power (FRAP), 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) activities, and inhibitory effect of α-glucosidase activity decreased in the small intestinal phase. Indigenous fruits or freeze-dried powders with Cy-3-Sa can be a better source of anthocyanin than Cy-3-G due to higher bioaccessibility in the small intestinal phase.

Process for production of microencapsulated anthocyanin pigments from Rosa pimpinellifolia L. fruits: optimization of aqueous two-phase extraction, microencapsulation by spray and freeze-drying, and storage stability evaluation

2020 ◽  
Vol 16 (9) ◽  
Author(s):  
Halil İbrahim Odabaş ◽  
Ilkay Koca
Keyword(s):  
Freeze Drying ◽  
Fold Increase ◽  
Drying Methods ◽  
Phase Extraction ◽  
Extraction Temperature ◽  
Two Phase ◽  
Freeze Dried ◽  
Anthocyanin Pigments ◽  
Promising Source ◽  
Aqueous Two Phase Extraction

AbstractRosa pimpinellifolia L. fruits (RPF) are promising source of anthocyanin pigments. The objectives of this study were to optimization of the aqueous two-phase extraction (ATPE) process of anthocyanin from RPF and microencapsulation of anthocyanin-rich RPF extract. The optimal ATPE conditions were as follows: 0% HCl, 30% ethanol, 19% ammonium sulfate, and liquid to solid ratio 51.71, 97.71 min, and 30°C extraction temperature. Predicted anthocyanin yield at the optimum conditions was 1578.90 mg cyanidin 3-glucoside equivalent/100 g dry fruit. ATPE resulting in 1.80-fold increase in the purity of anthocyanins when compared to conventional solvent extraction (CSE). The composition of the anthocyanins were determined with HPLC-QTOF-MS. Freeze-drying and spray-drying methods were employed for the production of microencapsulated anthocyanin pigments. The half times of microencapsulated anthocyanins at 4, 25 and 37°C were determined as 12.16, 6.60 and 3.12 months for freeze-dried microcapsules, and 16.50, 9.24 and 4.29 months for spray-dried microcapsules, respectively.

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