In vitro production of porcine zygotes using intracytoplasmic injection of vitrified sperm

The objectives of this study were to evaluate if vitrified porcine spermatozoa are able to maintain their capacity to produce zygotes in vitro using intracytoplasmic sperm injection (ICSI) and to evaluate the zygote development in two in vitro atmospheric conditions: 5% CO₂ and tri‐gas. A group of porcine oocytes maturated in vitro were injected with vitrified‐warmed sperm (treatment group) and another group, with sperm diluted and conserved at 17°C (control group). To evidence parthenogenetic activation, some oocytes were submitted to a Sham test. The injected oocytes were cultured in G1 medium at 38°C, 100% humidity and 5% CO₂ or tri‐gas. No significant differences (p > .05) were observed in embryo development between the oocytes injected with vitrified‐warmed sperm (31.8%; 36/113), and those injected with semen diluted and conserved at 17°C (35.5%; 32/90), when cultured in 5% CO₂ or under tri‐gas atmosphere (42.9%; 39/91 vs. 34.2%; 26/76, respectively). No significant differences (p > .05) were observed in the percentage of pronuclei (PN) obtained between 5% CO₂ and tri‐gas, within each treatment either. Of the 52 oocytes submitted to the Sham test, only two presented a female PN (activation) indicating that the PN observed in the treatment group were a product of fertilization and not parthenogenetic activation. To conclude, porcine sperm vitrified using spheres, at a concentration of 5 × 10⁶ spermatozoa/ml in TALP medium with 1% bovine serum albumin (BSA), conserve condensed and intact chromatin capable of producing early embryo development up to the pronuclear stage.     

Vitrification for bovine embryos with low‐quality grade

This study was conducted to examine the utility of vitrification for bovine embryos with low‐quality grade, and simple cryoprotectants dilution method for practitioners. In Experiment 1, survival of frozen embryos was compared with that of vitrified embryos using minimum volume cooling (MVC). Then, vitrified embryos were used to confirm the optimum sucrose concentration in Experiment 2. The survival rates of embryos that had been vitrified following diluted cryoprotectants with the one‐step in‐straw method were compared with those of fresh control embryos in Experiment 3. Frozen‐thawed or vitrified‐warmed blastocysts were cultured with TCM‐199 supplemented with 100 μmol/L beta‐mercaptoethanol +5% fetal bovine serum at 38.5°C in an atmosphere of 5% CO₂ in air, their survival after 24 hr were compared. The development to term of fair quality in vivo embryos after vitrification was examined in Experiment 4. Results show that survival rates of frozen‐thawed embryos were lower (p < .05) than that of vitrified‐warmed ones. When vitrified embryos were warmed in 0.3 mol/L sucrose in straws, their survival rate was 100%. The total cell numbers of vitrified‐warmed embryos were comparable to those of fresh control embryos. The six calves from 13 vitrified embryos were delivered in Experiment 4. These results indicate that MVC vitrification following one‐step cryoprotectants dilution is utilized to preserve low‐quality bovine embryos.     

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

The aim of this study was to determine the most efficient vitrification protocol for the cryopreservation of day 7 in vitro produced (IVP) porcine blastocysts. The post‐warm survival rate of blastocysts vitrified in control (17% dimethyl sulfoxide + 17% ethylene glycol [EG] + 0.4 mol/L sucrose) and commercial media did not differ, nor did the post‐warm survival rate of blastocysts vitrified in medium containing 1,2‐propandiol in place of EG. However, vitrifying embryos in EG alone decreased the cryosurvival rate (55.6% and 33.6%, respectively, p < .05). Furthermore, the post‐warm survival rates of blastocysts vitrified with either trehalose or sucrose as the non‐penetrating cryoprotectant did not differ. There was also no significant difference in post‐warm survival of blastocysts vitrified in control (38°C) media and room temperature (22°C) media with extended equilibration times, although when blastocysts were vitrified using control media at room temperature, the post‐warm survival rate increased (56.8%, 57.3%, 72.5%, respectively, p < .05). The findings show that most cryoprotectant combinations examined proved equally effective at supporting the post‐warm survival of IVP porcine blastocysts. The improved post‐warm survival rate of blastocysts vitrified using media held at room temperature suggests that the cryoprotectant toxicity exerted in 22°C media was reduced.     

Insulin‐like growth factor I enhances the developmental competence of yak embryos by modulating aquaporin 3

The objective of our present study was to determine the effects of insulin‐like growth factor I (IGF‐I) on the development of yak (Bos grunniens) embryos after cumulus–oocyte complex (COC) vitrification and warming followed by in vitro fertilization (IVF). In Experiment 1, the yak COCs underwent vitrification and then IVF. Embryos were incubated in synthetic oviductal fluid (SOF) supplemented with four concentrations (0, 50, 100 and 200 ng/ml) of IGF‐I, while the yak COCs without vitrification or IGF‐I supplementation acted as the control group; the BAX, BCL‐2, AQP3mRNA and aquaporin 3 (AQP3) protein expression levels in the five groups of blastocysts were evaluated using quantitative real‐time PCR and immunofluorescence analyses. In Experiment 2, the groups described above were fertilized and incubated. The cleavage rate, blastocyst rate, total cell count per blastocyst and the rate of growth of the inner cell mass (ICM) and trophectoderm (TE) were evaluated. The results were as follows: (1) the AQP3 gene expression and protein expression in the control and 100 ng/ml IGF‐I treatment groups were the highest. (2) The BAX gene expression was the lowest and the BCL‐2 gene expression was the highest in the control and 100 ng/ml IGF‐I treatment groups. (3) The rates of cleavage and blastocysts in the control and 100 ng/ml IGF‐I groups were higher than those in the other three groups. The total cell count per blastocyst in the vitrified and warmed 100 ng/ml IGF‐I group (106.7 ± 4.9) and the control group (107.3 ± 4.2) was higher than that in the vitrified and warmed 0 ng/ml IGF‐I (91.2 ± 3.1), 50 ng/ml IGF‐I (92.3 ± 3.7) and 200 ng/ml IGF‐I (92.4 ± 3.7) groups. Therefore, we conclude that IGF‐I can improve yak blastocyst developmental ability, cytomembrane permeability and formation of the blastocyst cavity after COC vitrification by improving the BAX, BCL‐2 and AQP3 expression levels.     

Calf production from vitrified bovine sexed embryos following in‐straw dilution

The objective of this study was to develop an in‐straw dilution method suitable for direct transfer of vitrified bovine sexed embryos. Embryo sexing was performed by molecular diagnosis. Several sexed and vitrified‐warmed embryos were transferred after evaluation of morphologically embryonic survival at warming and in‐straw dilution (Evaluation group). The other embryos were immediately directly transferred to recipients without first being expelled from the straws after in‐straw dilution (Non‐evaluation group). The pregnancy rates of vitrified sexed embryos were 38.7% and 34.8% in the Evaluation group and Non‐evaluation group, respectively, which were not significantly different. The viability of lower quality embryos before vitrification tended to be lower (P = 0.087) than that of the higher quality embryos regardless of evaluating embryos after warming and in‐straw dilution. The abortion rates were similar, and there was no difference between the two groups (13.9% and 12.5%, respectively). These results demonstrate that vitrified bovine sexed embryos can be vitrified and diluted by the in‐straw method and that the vitrified and warmed sexed embryos can develop to term.     

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.     

Expressions of cytochrome P450 aromatase and anti-Müllerian hormone in testes of fattening pigs by the timing of the first vaccination for immunocastration

In practice, two injections of gonadotropin-releasing hormone (GnRH) vaccine are recommended for pig immunocastration for effective outcomes. The present study aimed to investigate the expressions of cytochrome P450 aromatase (P450_arom) and anti-Müllerian hormone (AMH) in testes, testicular length and testicular histomorphometry of the fattening pigs receiving the first injection of GnRH vaccine 6 weeks earlier than the standard protocol. Based on vaccination protocol, 24 pigs were equally divided into three groups: T1 was vaccinated at 15 and 19 weeks of age, T2 received vaccine at 9 and 19 weeks of age and C remained intact. P450_arom and AMH expressions were analysed using immunohistochemistry and Western blot. The results revealed that testicular length was highest in C pigs, but not different between T1 and T2 groups (6.5 ± 0.2 versus 6.9 ± 0.3 cm, p = .538). Histomorphometry demonstrated that the height of spermatogenic epithelia, the diameter of seminiferous tubules and the number of seminiferous tubules between T1 and T2 groups were not different (p > .05). For P450_arom, immunohistochemistry revealed that H-score of C group was significantly higher than that of both T1 and T2 groups. Western blot analysis showed that C group possessed the densest protein band. Moreover, H-score between T1 and T2 groups was not significantly different. Protein band intensity between both groups was not apparently different. As for AMH, C pigs had significantly lower H-score than both T1 and T2 pigs. Furthermore, T2 pigs possessed significantly higher H-score than T1 pigs. Western blot analysis showed that the most intense protein band was found in T2 group. In summary, GnRH vaccine affected testicular development and functions. The first injection could be performed either at 9 or 15 weeks of age since both protocols contributed to comparable results in aspect of testicular length, histomorphometry and expressions of P450_arom and AMH.     

Successful Vitrification of In vivo Embryos Collected from Superovulated Japanese Black Cattle (Wagyu)

The aim of this study was to determine whether vitrification is an effective method when used for Japanese Black Cattle (Wagyu) in vivo‐derived embryos, collected following a superovulation treatment and embryo transfer (MOET) programme. In vivo‐derived morula and blastocysts collected on day 7 after artificial insemination, were vitrified using a modified droplet vitrification (MDV) procedure and subsequently warmed for transfer (ET) into synchronized recipients. Fresh embryos, and embryos cryopreserved using a standardized slow freezing procedure (direct thaw/direct transfer, DT) served as ET controls. Two different follicle‐stimulating hormone (FSH) sources, Folltropin^® Canada (FSH BAH, 24 donors) and a brand prepared by the Chinese Academy of Science (FSH CAS, 16 donors), were compared in a series of superovulation outcomes following well‐established FSH administration protocols. Following data analysis, the total number of ovulations recorded at the time of embryo flushing (10.5 vs 8.5; p = 0.28) and the total number of transferable embryos (6.2 vs 5.1; p = 0.52) were similar between the two FSH sources. ET for MDV (39.7%, n = 78), DT (35.2%, n = 71) and fresh controls (47.1%, n = 34) resulted in similar pregnancy rates (p > 0.05). When MDV was used, a higher pregnancy rate (42.6%) resulted from the transfer of vitrified morulae, when compared to the DT counterparts (24.3%), (p = 0.05). Transfer of vitrified morulae resulted also in higher pregnancy rate, when compared to the transfer of vitrified blastocysts (42.6% vs. 29.4%; p < 0.05). Transfer of DT blastocysts resulted in higher pregnancy rate than morulae, similarly cryopreserved (47.1% vs. 24.3%, p < 0.05). In conclusion, MDV is an effective alternative methodology for cryopreservation of in vivo‐derived embryos. This study gives also indication that, compared to vitrified blastocysts, MDV of morula stage embryos results in higher pregnancy rates following warming and transfer into synchronized recipients.     

Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7 µM ionomycin in TCM 199 medium (5 min) followed by 2 mM 6-DMAP in TCM 199 supplemented with 10% FBS (3 hr), then cultured and evaluated every 24 hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.     

Bax and Bcl‐2 are involved in the apoptosis induced by local testicular heating in the boar testis

The aim of this study was to determine whether the effect of Bax and Bcl‐2 on the apoptosis of germ cells is caused by local testicular heating (42°C, 1 hr) in boar testis. The testes of three boars were exposed to 42°C for 1 hr. Three other boars were assigned as control (no heat treatment). After 6 hr of heat treatment, all boars were castrated and the testes were harvested. Immunohistochemical results showed that a redistribution of Bax was caused by heat stress, and Bcl‐2 was expressed in the cytoplasm and nucleus. Western blot analyses and quantitative real‐time polymerase chain reaction (QRT‐PCR) showed that the protein and mRNA levels of Bax and Bcl‐2 were increased after local testicular heating. The number of TUNEL‐positive cells was increased in the seminiferous tubules compared with the control after local testicular heating. These results suggested that local testicular heating induced the apoptosis of germ cells by regulating the Bax and Bcl‐2 protein levels.     

Cell Counts and Survival to Vitrification of Bovine In Vitro Produced Blastocysts Subjected to Sublethal High Hydrostatic Pressure

This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp‐70) were also examined. Day 7 and 8 bovine in vitro‐produced blastocysts were submitted to an HHP treatment (60 MPa, at 32°C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post‐warming) and hatching (48 h post‐warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP‐treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32°C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP‐treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp‐70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post‐warming.     

Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes

Despite the numerous potential applications of oocyte cryopreservation, the poor success rate has limited its practical applications. In livestock, particularly in ovine, the oocytes have low developmental competence following vitrification/warming process. Considering the occurrence of osmotic and oxidative stresses during the vitrification/warming process, the application of antioxidants and osmolytes may improve the developmental competence of vitrified/warmed oocytes. In the present study, we aimed to evaluate the effects of the addition of ascorbic acid (AA) and N‐acetyl cysteine (NAC) as antioxidants and glycine as an organic osmolyte either to the vitrification/warming solutions (VWS) or to the IVM medium on the developmental competence of vitrified/warmed ovine germinal vesicle stage oocytes. The survival rate in the vitrified groups was significantly lower than fresh ones. In vitrified/warmed oocytes, there was no significant difference in survival rate between supplemented and non‐supplemented groups. The addition of AA and/or NAC to the VWS or IVM medium and adding glycine to the IVM medium reduced the proportion of apoptotic oocytes and fragmented embryos, which was reflected as an increase in the proportions of metaphase II stage oocytes and blastocyst production. The best result was achieved by supplementing the IVM medium with NAC. In our study condition, antioxidants and glycine could improve the developmental competence of vitrified/warmed ovine immature oocytes, especially when added during IVM.     

Vitrification of canine ovarian tissue using the Ovarian Tissue Cryosystem (OTC) device

The present study evaluated the effect of Ovarian Tissue Cryosystem (OTC) on follicular morphology and density, as well as on stromal cell density of vitrified canine ovarian tissue. Canine ovarian fragments collected from adult female dogs in stages of the random oestrous cycle were fixed (FC, fresh control) or vitrified (VIT) with an OTC device. After vitrification and warming, the fragments were fixed for histological analysis. Overall, the mean percentage of normal pre-antral follicles decreased after vitrification procedure (FC: 74.5% ± 1.6% vs. VIT: 52.05% ± 1.5%). Although the rates of normal primordial (71.1% ± 1.8%) and secondary (0.7% ± 0.4%) follicles vitrified showed a reduction (p < .05), vitrification using OTC showed considerable preservation of follicles, when compared to the fresh control (81.1% ± 1.5% and 2.3% ± 0.6%, respectively). The mean follicular density was maintained after vitrification (FC: 199.65 ± 12.8 vs. VIT: 199.68 ± 10.8), whereas the stromal cell density decreased in the VIT group. Based on the results, we recommend the use of OTC for vitrification of canine ovarian tissue.     

Cryopreservation of immature oocytes of the indigeneous Vietnamese Ban Pig

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus‐oocyte complexes were collected from antral follicles of 7–8 mo old female cyclic Ban pigs and vitrified in micro‐drops. Oocyte morphology, lipid content, post‐warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post‐warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo‐tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.     

Effect of Heat‐treatment on Accuracy of Infrared Spectroscopy and Digital and Optical Brix Refractometers for Measuring Immunoglobulin G Concentration in Bovine Colostrum

Background

Heat‐treatment of colostrum is a method developed to reduce calf exposure to pathogens. Infrared (IR) spectroscopy and Brix refractometers can be used for measuring colostral IgG concentration and assessing colostrum quality.

Objectives

To determine the impact of heat‐treatment on accuracy of IR spectroscopy and Brix refractometers for measuring colostral IgG concentration and assessing colostrum quality before and after heat‐treatment.

Animals

A total of 60 Holstein dairy cows on 8 commercial dairy farms.

Methods

A cross‐sectional study was designed to determine the effect of heat‐treatment at 60°C and 63°C each for 30 and 60 minutes duration on colostral IgG concentration measured by the reference radial immunodiffusion (RID) assay, IR spectroscopy, and digital and optical refractometers.

Results

Colostrum IgG concentration significantly decreased after heat‐treatment at 63°C for 30 or 60 minutes as measured by RID, but the IgG values remained unchanged when measured by IR spectroscopy and refractometers. The lowest correlation coefficient found between IR spectroscopy (r = 0.70) and RID results was in colostrum heat‐treated at 63°C for 60 minutes. For digital (r = 0.48) and optical (r = 0.50) refractometers, the lowest correlation coefficient was at 63°C for 30 minutes when compared to RID. The accuracy of the IR spectroscopy, digital and optical Brix refractometers was decreased from 91.7 to 80%, 81.7 to 45%, and 80 to 45%, respectively, when colostrum heat‐treated at 63°C for 60 minutes.

Conclusions and Clinical Importance

Radial immunodiffusion, IR spectroscopy, and Brix refractometers exhibit utility for measuring IgG concentration when colostrum heat‐treated at 60°C but does not detect decrease IgG concentrations when heat‐treated at 63°C.     

Solid‐Surface Vitrification and In‐Straw Dilution After Warming of In Vitro‐Produced Bovine Embryos

Three experiments were designed to test a solid‐surface vitrification system for bovine in vitro‐produced embryos and to develop a simple method of in‐straw dilution after warming, which can be potentially used for direct transfer in the field. Experiment 1 evaluated embryo survival rates (i.e. re‐expansion and hatching) after vitrification and warming in three different solutions: VS1 (20% ethylene glycol (EG) + 20% propanediol (PROH) + 0.25 m trehalose (Tr)), VS2 (20% EG + 1M Tr) or VS3 (30% EG + 0.75 m Tr). Re‐expansion and hatching rates were higher (p < 0.05) for embryos vitrified in VS3 (72.2 ± 1.9 and 58.2 ± 0.8) than VS1 (64.4 ± 0.9 and 37.2 ± 2.5) or VS2 (68.5 ± 1.5 and 49.6 ± 1.0; p < 0.05). Experiment 2 was designed to compare two methods of vitrification: glass micropipettes or solid surface, using the VS1 or VS3 solutions. No significant differences were detected between the two methods; but re‐expansion and hatching rates were higher (p < 0.05) with VS3 (73.5 ± 3.1 and 47.1 ± 2.1) than VS1 (63.3 ± 3.3 and 39.7 ± 2.8). In experiment 3, embryos were vitrified by solid surface in VS1 or VS3 solutions and cryoprotectants were diluted in‐straw after warming in a TCM 199, 0.25 m sucrose solution or holding media. Survival rates of embryos vitrified in VS3 did not differ between those exposed to 0.25 m sucrose (74.7 ± 1.3 and 57.2 ± 2.2) or holding (77.3 ± 1.4 and 58.0 ± 2.5) medium after warming; however, survival rates of embryos vitrified in VS1 were higher (p < 0.05) in those exposed to 0.25 m sucrose (67.7 ± 2.3 and 47.0 ± 1.7) than holding medium (54.5 ± 1.0 and 27.7 ± 3.1). In conclusion, solid‐surface vitrification using simplified EG‐based solutions and in‐straw dilution with holding media may be a practical alternative for cryopreservation and direct transfer of in vitro‐produced bovine embryos.     

Notoginsenoside R1 protects boar sperm during liquid storage at 17°C

Reactive oxygen species (ROS) damage mammalian sperm during liquid storage. Notoginsenoside R1 (NR1) is a compound isolated from the roots of Panax notoginseng; it has powerful ROS-scavenging activities. This work hypothesized that the antioxidant capacity of NR1 could improve boar sperm quality and fertility during liquid storage. During liquid storage at 17°C, the supplementation of semen extender with NR1 (50 μM) significantly improved sperm motility, membrane integrity and acrosome integrity after 5 days of preservation. NR1 treatment also reduced ROS and lipid peroxidation (LPO) levels at day 5 (p <0.05). Higher glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) levels and sperm–zona pellucida binding capacity were observed in the 50 μM NR1 group than those in the control group at day 7 (p <0.05). Importantly, statistical analysis of the fertility of 200 sows indicated that addition of NR1 to the extender improved the fertility parameters of boar spermatozoa during liquid storage at 17°C (p <0.05). These results demonstrate the practical feasibility of using 50 μM NR1 as an antioxidant in boar extender during liquid storage at 17°C, which is beneficial to both spermatozoa quality and fertility.     

Effect of Meiotic Stages During <Emphasis Type="Italic">In Vitro</Emphasis> Maturation on the Survival of Vitrified-Warmed Buffalo Oocytes

The present study was conducted to investigate the effect of meiotic stages during in vitro maturation (IVM) on the survival of vitrified-warmed buffalo oocytes, vitrified at different stages of IVM. Cumulus oocyte complexes obtained from slaughterhouse ovaries were randomly divided into 6 groups: control (non-vitrified, matured for 24 h at 38 ± 1^°C, 5% CO₂ in humidified air), and those matured for 0 h (vitrified before IVM) or 6, 12, 18 and 24 h before vitrification. Cumulus oocyte complexes were vitrified in solution consisting of 40% w/v propylene glycol and 0.25 mol/L trehalose in phosphate-buffered saline supplemented with 4% w/v bovine serum albumin. Vitrified cumulus oocyte complexes were stored at −196^∘C (liquid nitrogen) for at least 7 days and then thawed at 37^°C; cryoprotectant was removed with 1 mol/L sucrose solution. Cumulus oocyte complexes in the 0, 6, 12, 18 and 24 h groups were then matured for an additional 24, 18, 12, 6 and 0 h, respectively, to complete 24 h of IVM. Among the five vitrification groups, 89–92% of cumulus oocyte complexes were recovered, after warming, of which 84–91% were morphologically normal. Overall survivability of vitrified cumulus oocyte complexes was lower (p < 0.05) than that of non-vitrified cumulus oocyte complexes (94.5%). Survival rates of cumulus oocyte complexes matured 24 h prior to vitrification (61.3%) were higher (p < 0.05) than those matured for 12 h (46.7%), 6 h (40.6%) and 0 h (37.6%). Nuclear status following 24 h IVM was assessed. A higher proportion of non-vitrified (control) oocytes (72.7%) reached metaphase II (M-II) stage in control than oocytes vitrified for 24 h (60.0%), 18 h (54.4), 12 h (42.3%), 6 h (33.3%) and 0 h (31.6%) (p < 0.05). The results suggest that length of time in maturation medium prior to vitrification influences post-thaw survivability of buffalo oocytes; longer intervals resulted in higher survival rates.     

Effect of permeable cryoprotectant‐free vitrification on DNA fragmentation of equine oocyte–cumulus cells

DNA fragmentation of cumulus cells could be used as an indicator of oocyte vitrification success as an indirect indicator of the quality of the oocyte. This study was designed to compare the DNA fragmentation of post‐mortem equine cumulus cells before or after vitrification in the absence of permeable cryoprotectant agents. Cumulus–oocyte complexes (COCs; n = 56) were recovered from slaughterhouse ovaries and subjected to in vitro maturation (42 hr/38.2°C/5%CO₂) before (control group) or after a permeable cryoprotectant‐free vitrification method using 1 M sucrose (vitrification group). After in vitro maturation, COCs were denuded, and cumulus cells were washed and stored at ?80°C until thawing. Cumulus cell samples were processed with the chromatin dispersion test (Ovoselect, Halotech DNA, Spain). Low, high and total DNA fragmentation percentages of cumulus cells were recorded and compared between the two groups by Student's t test. Results were expressed as mean ± SEM. The vitrified group resulted in significantly higher (p < 0.05) percentages for low (16.81 ± 1.62 vs. 6.63 ± 0.77) and total (21.14 ± 1.84 vs. 12.76 ± 1.48) DNA fragmentation of cumulus cells. There were no significant differences between groups for high DNA fragmentation of cumulus cells. In conclusion, permeable cryoprotectant‐free vitrification of equine oocytes increased the total DNA fragmentation rate of cumulus cells but protected them against high DNA fragmentation rates. Further studies are needed to examine the relationship between DNA fragmentation of cumulus cells and the developmental competence of equine oocytes.     

Granulosa cells in three-dimensional culture: A follicle-like structure for domestic cat vitrified oocytes

Follicle-like structures are three-dimensional matrices joint with living cells that allow the in vitro development of female gametes in more physiological conditions. They have been shown to be beneficial to fresh oocytes in different species, and in this study, domestic cat (Felis silvestris catus) granulosa cells were used to create a functional follicle-like structure aimed at supporting the in vitro maturation of conspecific vitrified oocytes, key players of fertility preservation programmes that usually struggle to acquire their full developmental competence after warming. Cat granulosa cells were cultured for up to 6 days in three-dimensional barium alginate microcapsules (i.e. follicle-like structures) or in two-dimensional monolayers, and their steroidogenic ability (estradiol and progesterone secretion) was assessed to confirm their functionality. The same systems were used (on day 2 or 6 of granulosa cells culture) for the in vitro maturation (IVM) of Cryotop^® vitrified immature cat oocytes and compared with microdrops of IVM medium without cells (control). Granulosa cells were able to maintain their functionality in vitro in both the conditions, even if with a different extent of hormonal secretion along culture (p = .02). Vitrified oocytes resumed meiosis at higher rates when cultured with 2 days old granulosa cells (p = .03), but full maturation rates slightly raised when granulosa cells were cultured longer, albeit without differences with the control group. This study paved the road for the creation of enriched culture systems in the domestic cat, but innovations are strongly needed for vitrified oocytes that deserve better chances to develop in vitro.