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Quan G.B.,Yunnan Animal Science and Veterinary Institute | Quan G.B.,Yunnan Provincial Meat Caprine Engineering Research Center | Quan G.B.,Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement | Wu G.Q.,Yunnan Animal Science and Veterinary Institute | And 12 more authors.
Cryobiology | Year: 2015

In order to assess effects of vitrification on ovine oocytes at the germinal vesicle (GV) stage, the conventional plastic straw (CS), the open-pulled straw (OPS), and Cryoloop were used to vitrify ovine oocytes. Oocytes were randomly divided into five groups: (1) Control; (2) Oocytes exposed to vitrification and dilution solutions without any cryopreservation (toxicity); (3) Oocytes vitrified using CS (CS); (4) Oocytes vitrified using OPS (OPS), and (5) Oocytes vitrified using Cryoloop (Cryoloop). The viability, cumulus cell expansion, nuclear maturation after in vitro maturation (IVM), and developmental capability of vitrified oocytes following parthenogenetic activation (PA) or in vitro fertilization (IVF) were assessed. The pretreatment in the vitrification and dilution solutions without any freezing or thawing did not adversely influence oocytes. The viability of vitrified oocytes were significantly declined compared to unfrozen oocytes (P < 0.05). The viability of oocytes vitrified using open-pulled straws or Cryoloop was significantly higher than that in the CS group (P < 0.05). After IVM, the percentage of oocytes reaching to the metaphase II (MII) stage was significantly higher with Cryoloop and OPS following by CS. However, the in vitro maturing percentage of vitrified oocytes was significantly less than that of unfrozen oocytes (P < 0.05). After PA, the developmental capability of vitrified oocytes was significantly decreased compared to unfrozen oocytes. The cleavage rate of oocytes vitrified using conventional plastic straws was significantly less than those of the other freezing groups (P < 0.05). The cleaving capability of oocytes vitrified using Cryoloop was significantly increased compared to the OPS group. However, there was no significant difference existing amongst the freezing groups as concerning the blastocyst rate. Following IVF, the developmental capability of vitrified oocytes was severely damaged compared to that of unfrozen oocytes. The cleavage rate of oocytes vitrified with Cryoloop was similar to that of oocytes vitrified with open-pulled straws. However, the cleavage rate of vitrified oocytes in the CS group was significantly less than that in the OPS or Cryoloop group (P < 0.05). None of oocytes vitrified using conventional plastic straw developed to the blastocyst stage following IVF. There was no significant difference existing between OPS and Cryoloop with respect to the blastocyst rate. After staining with cFDA and PI, cumulus cells surrounding oocytes were partly damaged by vitrification and thawing while the membrane of vitrified oocyte still remained intact. In conclusion, vitrification can seriously damage ovine immature oocytes and cumulus cells surrounding oocytes, which may subsequently affect their developmental capability. Finally, this study further proves that increasing the freezing and thawing velocity benefits survival of vitrified immature oocytes. © 2015. Source


Quan G.B.,Yunnan Animal Science and Veterinary Institute | Quan G.B.,Yunnan Provincial Meat Caprine Engineering Research Center | Quan G.B.,Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement | Wu G.Q.,Yunnan Animal Science and Veterinary Institute | And 12 more authors.
Small Ruminant Research | Year: 2016

Cryopreservation can seriously damage structure and physiological function of mammalian spermatozoa. However, liquid storage at reduced temperature may be an alternative of cryopreservation if artificial insemination is performed in relatively short time. In this study, effects of the Tris-hydroxymethyl aminomethane (Tris), N-Tris (hydroxymethyl) -methylaminoethane-sulfonic acid (Tes), or skim milk based extender on ram spermatozoa during liquid storage at 4 °C were assessed and compared. Semen samples from 6 adult Yunnan semi-fine wool rams with proven fertility were pooled, equally divided into 3 groups according to extenders, and preserved at 4 °C for various durations (0, 1, 3, 5, and 7 days). The motility, moving velocity, and hypo-osmotic swelling capability of spermatozoa were evaluated using a computer assisted sperm analyzer system. The acrosome status, membrane integrity, distribution of phosphatidylserine (PS), and mitochondrial membrane potential (MMP) were analyzed with flow cytometry. The results indicated that following liquid storage in the Tris or Tes based extender for 3 days, the motility, hypo-osmotic swelling capability, and MMP of spermatozoa did not show a significant decrease. Additionally, liquid storage in the Tris or Tes based extender for 24 h did not deleteriously affect the moving velocity, acrosome status, and membrane integrity of spermatozoa. However, after liquid storage for 24 h, the percentage of spermatozoa with membrane exposed PS was significantly increased. On the contrary, the negative effects of skim milk on the above parameters of ram spermatozoa became more serious compared to the Tris or Tes based extender after liquid storage for 24 h (P< 0.05). There was no significant difference existing between the Tris and Tes based extender as concerning the parameters evaluated in this study. In conclusion, the Tris or Tes based extender may be more suitable for liquid storage of ram spermatozoa at reduced temperature compared to the skim milk based extender. Tris can be substituted by Tes for liquid storage of ram semen. The future study should focus on assessment of molecular changes and fertility of ram spermatozoa after liquid storage. © 2015 Elsevier B.V. Source


Quan G.B.,Yunnan Animal Science and Veterinary Institute | Quan G.B.,Yunnan Provincial Meat Caprine Engineering Research Center | Quan G.B.,Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement | Ma Y.,Yunnan Animal Science and Veterinary Institute | And 21 more authors.
Cryobiology | Year: 2015

Cytometric sorting of frozen-thawed sperm can overcome difficulties caused by the unavailability of sorting facilities on farms where semen is collected from male livestock. In order to optimize the cytometric sex-sorting procedure, effects of Hoechst33342 staining on the viability and cytometric sorting efficiency of frozen-thawed ram sperm were evaluated. The frozen-thawed sperm were stained with Hoechst33342 at various dye concentrations (80. μM, 120. μM, 160. μM, 200. μM, 240. μM, or 320. μM) for 45. min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160. μM Hoechst33342 for various durations (0. min, 15. min, 30. min, 45. min, 60. min, 75. min, or 90. min) to evaluate effects of staining duration. Sperm motility and moving velocity were analyzed using a computer-assisted sperm analysis system (CASAS). Acrosome status, membrane integrity, and distribution of phosphatidylserine (PS) in Hoechst33342-stained sperm were analyzed using flow cytometry after staining with fluorescein isothiocyanate-labeled lectin from pisum sativum (FITC-PSA), Annexin V, or propidium iodide (PI). The fertility of Hoechst33342-stained sperm was analyzed by in vitro fertilization (IVF). A high-speed cell sorter was used to evaluate effects of Hoechst33342 staining on cytometric sex-sorting of frozen-thawed sperm. The motility, moving velocity, membrane integrity, and PS distribution of Hoechst33342-stained sperm were significantly different from that of immediately thawed sperm (P<. 0.05). However, there is no significant difference existing among the Hoechst33342-stained groups with respect to the above evaluated parameters. Additionally, along with the staining durations, the adverse effects of the staining procedure on sperm showed a steady increase. However, Hoechst33342 staining did not damage acrosome and in vitro fertilizing capability of frozen-thawed ram sperm. Results of cytometric sorting indicated that frozen-thawed sperm can be efficiently sorted into two sperm populations with X and Y chromosome when the Hoechst33342 concentration was 160. μM. Moreover, when the staining duration was equal to or longer than 45. min, the frozen-thawed sperm can be successfully sorted in the presence of 160. μM Hoechst33342. In conclusion, Hoechst33342 staining can detrimentally influence viability of frozen-thawed ram sperm except acrosome and in vitro fertilizing capability. Accordingly, the minimum values of Hoechst33342 concentration and staining duration can be set at 160. μM and 45. min respectively. However, the maximum values of Hoechst33342 concentration and staining duration were not determined based on the current study. Further research on how to reduce injuries caused by freezing, thawing, and Hoechst33342 staining on frozen-thawed ram sperm is needed. © 2014 Elsevier Inc. Source

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