Yunnan Provincial Meat Caprine Engineering Research Center

Kunming, China

Yunnan Provincial Meat Caprine Engineering Research Center

Kunming, China

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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.


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.


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 | Li D.J.,Yunnan Animal Science and Veterinary Institute | And 14 more authors.
Small Ruminant Research | Year: 2015

The effects of cyclohexanhexol-derived synthetic ice blockers (SIBs) including 1,3-cyclohexanediol (1,3-CHD) and 1,4-cyclohexanediol (1,4-CHD) on chilled or frozen Yunnan semi-fine wool ram spermatozoa were systematically assessed and compared in this study. The collected semen was pooled and equally divided into two parts. In the first part, in order to evaluate the effect of SIBs on chilled spermatozoa before freezing, the semen was divided into nine groups according to the concentrations of SIBs (0, 25. mM, 50. mM, 100. mM, or 200. mM). In the second part, the treatment of semen was similar to the first part. However, except for the cooling and equilibrating procedures, spermatozoa were preserved in liquid nitrogen for one month. Motility, moving velocity, acrosome status, membrane integrity, and mitochondrial membrane potential (MMP) of frozen/thawed spermatozoa were determined using a computer-assisted spermatozoa analysis system (CASAS), fluorescence staining, and flow cytometry. The data indicated that when the concentrations of SIBs were less than or equal to 100. mM, there is no significant difference among the treating groups as concern spermatozoa motility and moving velocity following equilibration at 5. °C. However, when the concentrations of SIBs were increased to 200. mM, the motility and moving velocity of chilled ram spermatozoa were significantly decreased compared to the control spermatozoa ( P<. 0.05). With elevation of the SIB concentrations, the motility of frozen/thawed spermatozoa was firstly increased and then reduced. With a concentration of 50. mM or 100. mM, the spermatozoa motility was approximately 50% and significantly more than the other groups ( P<. 0.05). Additionally, when the SIB concentrations were less than or equal to 100. mM, there is no significant difference between the treating groups as concern moving velocity. Moreover, when the concentration of 1,3-CHD or 1,4-CHD was 100. mM, the percentage of frozen/thawed ram spermatozoa with intact acrosome was 82.57. ±. 8.97% and 79.51. ±. 13.49% respectively, which were significantly higher than that of the control spermatozoa (70.21. ±. 13.24%, P<. 0.05). Additionally, with elevation of the SIB concentrations, the percentages of frozen/thawed ram spermatozoa with damaged membrane showed a steady increase. Finally, the presence of 100. mM SIBs can significantly improve the MMP status of frozen/thawed ram spermatozoa compared to spermatozoa frozen in the absence of SIBs. In conclusion, SIBs can improve the viability of frozen/thawed ram spermatozoa as concern motility, acrosome status, and MMP. However, SIBs cannot improve moving velocity and membrane integrity of frozen/thawed spermatozoa. Finally, the protective effect of 1,3-CHD is not superior to that of 1,4-CHD on chilled or frozen ram spermatozoa. © 2014 Elsevier B.V.


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.


Wu G.Q.,Yunnan Animal Science and Veterinary Institute | Wu G.Q.,Yunnan Prov Engineering Laboratory Of Animal Genetic Resource Conservation And Germplasm Enhancement | Wu G.Q.,Yunnan Provincial Meat Caprine Engineering Research Center | Lv C.R.,Yunnan Animal Science and Veterinary Institute | And 17 more authors.
Biopreservation and Biobanking | Year: 2016

In this study, the protective effects of monosaccharides (glucose and fructose) and sugar alcohols (mannitol, sorbitol, and xylitol) on frozen ram spermatozoa were evaluated and compared. The motility, moving velocity, and hypoosmotic swelling capability of spermatozoa frozen with monosaccharide or sugar alcohol were measured using a computer-assisted spermatozoa analyzer system. The acrosome status, membrane integrity, distribution of phosphatidylserine (PS), and mitochondrial membrane potential (MMP) were analyzed using fluorescence staining and flow cytometry. The results indicated that similar to glucose or fructose, the presence of sugar alcohol in the freezing extender cannot significantly improve the motility and moving velocity of ram spermatozoa equilibrated at 5°C. In terms of motility, pathway velocity, curve velocity, hypoosmotic swelling capability, acrosome and membrane integrity, and MMP, the inclusion of mannitol or sorbitol in the extender can significantly improve the quality of frozen-thawed ram spermatozoa compared to glucose or fructose. However, the effects of mannitol or sorbitol on linear velocity and PS distribution of frozen-thawed spermatozoa were similar to those of the monosaccharides (p > 0.05). In addition, the ability of xylitol to protect acrosome and maintain MMP in frozen-thawed spermatozoa was significantly higher compared with glucose or fructose (p < 0.05), although it could not improve the other evaluated parameters. Finally, there is no significant difference existing between mannitol and sorbitol with respect to the above evaluated parameters. In conclusion, the replacement of glucose or fructose by mannitol or sorbitol in a freezing extender can improve the postthaw quality of ram spermatozoa under specific freezing conditions. Moreover, the protective effects of mannitol and sorbitol on frozen-thawed ram spermatozoa are superior to that of xylitol. However, in the presence of sugar alcohols, the cryoinjury on spermatozoa membrane is still serious. In the future, the question of protecting the membrane of frozen-thawed spermatozoa needs further research. © Copyright 2016, Mary Ann Liebert, Inc. 2016.

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