Forced Collapse of the Blastocoel Cavity Improves Developmental Potential in Cryopreserved Bovine Blastocysts by Slow‐Rate Freezing and Vitrification

This study was conducted to evaluate the effectiveness of forced collapse of the blastocoel before slow‐rate freezing and vitrification of bovine blastocysts. Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production using the embryo transfer technique. However, the low efficiency of frozen–thawed embryos survival and further development is a crucial problem. In this study, bovine in vitro and in vivo blastocysts were slow‐rate frozen and vitrified after forced blastocoele collapse (FBC) of the blastocyst cavity by puncturing the blastocoele with a pulled Pasteur pipet. Differences in the developmental potential of frozen–thawed blastocysts derived from FBC and non‐FBC groups were found in both slow‐rate freezing and vitrification. Furthermore, we found that the total cell number of blastocysts in FBC groups was increased and the index of apoptosis in FBC groups was decreased. Consistent with these results, real‐time RT‐PCR analysis data showed that expression of the anti‐apoptotic Bcl‐XL gene was significantly increased by FBC groups, whereas expression of the pro‐apoptotic Bax gene was significantly decreased by FBC groups. Our results also showed that pregnancy outcomes in both slow‐rate frozen and vitrified bovine in vivo blastocysts could be improved by reducing the fluid content after FBC of the blastocyst cavity. Therefore, we suggest that FBC of the blastocyst cavity with a pulled Pasteur pipet is an effective pre‐treatment technique for both slow‐rate freezing and vitrification of bovine blastocysts.     

Cryopreservation and In Vitro culture of Preimplantation Embryos in Djungarian Hamster (Phodopus sungorus)

Although embryo cryobanking was applied to Syrian golden and to Campbell's hamsters, no attempt has been made at freezing embryos in Djungarian hamsters. Four‐cell stage embryos were flushed from the reproductive ducts of pregnant females before noon of the third‐day post coitum and frozen in 0.25‐ml straws according to standard procedures of slow cooling. A mixture of permeating (ethylene glycol) and non‐permeating (sucrose) cryoprotectants was used. The thawing was performed by incubating at RT for 40 s followed by 40 s in a water bath at 30.0°C. Most (66.7%) of the non‐frozen four‐cell embryos developed up to the morula stage in rat one‐cell embryo culture medium (R1ECM). The use of hamster embryo culture medium (HECM) yielded fewer morulas (18.2%) during the same 24‐h period of culture. The rate of embryo's surviving the freezing–thawing procedures, as estimated by light microscopy, was 60.7–68.8%. After 24‐h culturing in R1ECM, 64.7% of frozen–thawed four‐cell embryos developed and all of them reached the morula stage. Supplementation of R1ECM with GM‐CSF (2 ng/ml) improved the rate of Djungarian hamster frozen–thawed embryo development: 100% of the four‐cell stage embryos developed, 50% of them achieved the morula stage, and 50% developed even further and reached the blastocyst stage within 24 h of culturing. This study reports the world's first successful transfer of frozen–thawed Djungarian hamster embryos yielding term pups. Taken together, the results of this study demonstrate the possibility of applying some key reproductive technologies, that is, embryo freezing/cryopreservation and in vitro culture, to Djungarian hamsters.     

Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification

This study was carried out to compare the post‐thaw cryosurvival rate and the level of apoptosis in vitro produced zona‐free cloned buffalo blastocysts subjected to slow freezing or vitrification in open‐pulled straws (OPS). Zona‐free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re‐expansion rate following post‐thaw culture for 22–24 h. The post‐thaw re‐expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen‐thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non‐cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non‐cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona‐free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.     

Vitrification of Immature Feline Oocytes with a Commercial Kit for Bovine Embryo Vitrification

The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p < 0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p < 0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p < 0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture.     

Effect of Different Extenders on In Vitro Characteristics of Feline Epididymal Sperm During Cryopreservation

To evaluate and compare the efficacy of various extenders for the cryopreservation of epididymal cat spermatozoa, two experiments were planned. Bovine and equine commercial extenders in the experiment 1 and TRIS–egg yolk–based extenders in experiment 2 were separately studied since the number of sperm collected per cat is reduced. Epididymal sperm samples were packaged into 0.25‐ml straws and frozen. Vigour, motility, morphology, acrosome status, sperm viability and functional membrane integrity were assessed at collection, after cooling and after thawing, while DNA integrity was evaluated at 0‐ and 6‐h post‐thaw. Experiment 1 compared the effect of three non‐feline commercial extenders – based on TRIS–egg yolk (Triladyl), egg‐yolk‐free medium (AndroMed) and skimmed milk‐egg yolk (Gent) – on the quality of frozen‐thawed epididymal cat sperm. Values for sperm motility and functional membrane integrity in cooled sperm diluted in Triladyl were higher (p < 0.001) than those recorded for Andromed and Gent. Except sperm morphology, the other assessed characteristics showed significant higher values in frozen‐thawed sperm diluted in Triladyl than in Andromed and Gent extenders. Experiment 2 analysed the effects of three TRIS–egg yolk–based extenders, one non‐feline commercial (Triladyl) and the other two prepared using different monosaccharides (glucose and fructose), on freezing‐thawed sperm. Results showed that specifically prepared extenders for cryopreservation of feline spermatozoa performed better than the commercial extender Triladyl, although sperm quality during the freezing‐thawing process did not significantly differ associated with the type of monosaccharide (glucose vs fructose) added to the mentioned extenders. Although TRIS–egg yolk–based extenders prepared in experiment 2 improved sperm cryoprotection, Triladyl remains a good option for practitioners who, for ease of use and availability, prefer to work with commercial extenders.     

Preincubation with green tea polyphenol extract is beneficial for attenuating sperm injury caused by freezing‐thawing in swine

Polyphenols (PFs) extracted from green tea, known to be potent anti‐oxidants, have been reported to be effective in increasing the motility and viability of mammalian sperm, preserved in a liquid form. Therefore, we tested whether PFs might also be effective for maintaining the integrity of frozen‐thawed boar spermatozoa. Ejaculates, collected from Clawn miniature pigs, were diluted in a semen extender containing various amounts of PFs (0, 0.01, 0.05, 0.1 and 0.2% w/v) and then stored at 15°C overnight. The semen samples were processed, using the straw freezing procedure, and then frozen in liquid nitrogen. After rapid thawing at 40°C, the spermatozoa were subjected to several assays to evaluate semen quality. Spermatozoa frozen in a medium containing 0.01% w/v PFs exhibited significantly (P < 0.05) higher degrees of post‐thawed viability and acrosomal integrity than those stored in the absence of PFs. However, no change in the mitochondrial activity was noted between the two groups. The inclusion of 0.01% PFs in the semen extender was significantly (P < 0.05) effective in increasing both the rates of monospermic oocyte formation and of blastocyst formation. These findings indicate that preincubation with the semen extender, containing 0.01% PFs prior to freezing, exerts a protective effect on boar sperm by preventing injuries associated with freezing‐thawing.     

白藜芦醇对山羊冷冻精子质膜、DNA完整性和温度耐受性的影响

试验旨在研究白藜芦醇对山羊冷冻精子质膜、DNA完整性和温度耐受性的影响。采用假阴道法采集8只云上黑山羊精液,用含不同浓度（0、0.1、1、10和20 μmol//L）白藜芦醇的Optidyl稀释液稀释后进行细管分装,对照组不添加白藜芦醇。在5 ℃平衡4 h后,将细管于液氮蒸气中预冻10 min,最后在液氮中保存30 d。37 ℃水浴解冻后,采用低渗耐受性试验检测质膜完整性和温度耐受性,精子染色质扩散法检测DNA碎片率等指标。结果显示,10 μmol/L白藜芦醇冷冻组精子弯尾率显著高于其他各处理组（P<0.05）,而其他各冷冻组之间无显著性差异（P>0.05）;10 μmol/L白藜芦醇冷冻组精子DNA碎片率极显著高于鲜精组（P<0.01）,极显著低于未添加白藜芦醇冷冻组（P<0.01）。温度耐受性试验结果表明,不同浓度白藜芦醇冷冻组精子37 ℃水浴1 h后的精子弯尾率以10 μmol/L冷冻组最高,与其他各冷冻组间差异极显著（P<0.01）;精子弯尾率随着孵育时间的延长而呈逐步下降的趋势,当孵育4 h时,10 μmol/L白藜芦醇冷冻组精子弯尾率与对照组无显著差异（P>0.05）。透射电镜结果表明,10 μmol/L白藜芦醇组精子质膜完整率显著高于不添加白藜芦醇对照组（P<0.05）。上述结果表明,在冷冻稀释液中添加白藜芦醇可显著改善山羊冻精质膜状态、DNA完整率和温度耐受性,其最佳作用浓度为10 μmol/L,但白藜芦醇是否能改善山羊冻精的人工授精效果还有待进一步研究。     

Incidence and avoidance of zona fracture in cryopreserved bovine ova and embryos

Ova (n=62), which were collected from slaughterhouse bovine ovaries, and embryos (n=26), which were non-surgically recovered from 11 superovulated crossbred donor cows, were frozen. The frozen ova and embryos were then thawed using two conventional thawing protocols, i.e. at 37 °C for 30 seconds in a water bath and at 25 °C for 2 minutes in air. Some 64.5% of the ova and 53.8% of the embryos thawed in the water bath and 16.1% of the ova and 7.7% of the embryos thawed in ambient air exhibited fractured z:onae pellucidae. The slow thawing protocol had a lower incidence of zona damage in cryopreserved oval and embryos than the fast thawing protocol. A low pregnancy rate (12.5%) was recorded for embryos transferred with zona fracture while embryos transferred with intact zonae had a rate of 35.3%) indicating that embryos with zona damage are less viable.     

Incidence and avoidance of zona fracture in cryopreserved bovine ova and embryos

Ova (n=62), which were collected from slaughterhouse bovine ovaries, and embryos (n=26), which were non-surgically recovered from 11 superovulated crossbred donor cows, were frozen. The frozen ova and embryos were then thawed using two conventional thawing protocols, i.e. at 37 degrees C for 30 seconds in a water bath and at 25 degrees C for 2 minutes in air. Some 64.5% of the ova and 53.8% of the embryos thawed in the water bath and 16.1% of the ova and 7.7% of the embryos thawed in ambient air exhibited fractured zonae pellucidae. The slow thawing protocol had a lower incidence of zona damage in cryopreserved oval and embryos than the fast thawing protocol. A low pregnancy rate (12.5%) was recorded for embryos transferred with zona fracture while embryos transferred with intact zonae had a rate of 35.3%) indicating that embryos with zona damage are less viable.     

In vitro development of OPU‐derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen‐thawed embryos after transfer

The optimization of single‐embryo culture conditions is very important, particularly in the in vitro production of bovine embryos using the ovum pick‐up (OPU) procedure. The purpose of this study was to examine the development of embryos derived from oocytes obtained by OPU that were cultured either individually or in groups in medium supplemented with or without sericin and to investigate the viability of the frozen‐thawed embryos after a direct transfer. When two‐cell‐stage embryos were cultured either individually or in groups for 7 days in CR1aa medium supplemented with or without 0.5% sericin, the rates of development to blastocysts and freezable blastocysts were significantly lower for the embryos cultured individually without sericin than for the embryos cultured in groups with or without sericin. Moreover, the rate of development to freezable blastocysts of the embryos cultured individually with sericin was significantly higher than that of the embryos cultured without sericin. When the frozen‐thawed embryos were transferred directly to recipients, the rates of pregnancy, abortion, stillbirth and normal calving in the recipients were similar among the groups, irrespective of the culture conditions and sericin supplementation. Our findings indicate that supplementation with sericin during embryo culture improves the quality of the embryos cultured individually but not the viability of the frozen‐thawed embryos after transfer to recipients.     

Cryosurvival and pregnancy rates: One‐step protocol for freezing–thawing Shangri‐la Yak (Bos grunniens) Embryos

The yak is one of the most important and economically useful animals for highlanders. The decline in the yak population requires effective measures for the conservation and multiplication of elite germplasm. A standardized protocol will simplify the freezing and warming of yak embryos in straw and facilitate embryo transfer. In this work, we investigated a one‐step protocol that uses a stable basal medium, which comprised a warming medium (1.08 M sucrose) and a freezing medium (EFS40). We also assessed the effects of the new transfer method on embryo survival. A total of 145 yak frozen embryos were thawed in a standard medium system. The one‐step protocol led to a high recovery percentage (84.93) of yak embryos that survived vitrification and warming. The in vitro survival rates of these embryos significantly different from those of embryos frozen–thawed via the conventional method. The 95 embryos frozen–thawed via our one‐step protocol were then implanted in selected recipients. Thirty‐six singleton pregnancies were established. In conclusion, the proposed one‐step method is a simple, safe, and standardized freezing–thawing protocol that ensures embryo survival and quality under field conditions. This study establishes new possibilities for the widespread use of embryo transfer in yaks.     

Use of a rapid method of cryopreservation of bovine embryos for non-surgical transfer in transplantation practice

The embryos were frozen and thawed in Cassou minipaillette by a rapid method. Embryos with cryoprotective agent (glycerol, 1.5 M) were placed directly into the freezing medium at the temperature of -6 to -7 degrees C, frozen after seedling at the temperature decrease by 0.3 to 0.5 degrees C per minute to the temperature of -32 degrees C and then transferred directly into liquid nitrogen. They were thawed in a bath warm 20 to 37 degrees C. After thawed the cryoprotective agent was evacuated in 1.1 M sucrose. The best-quality embryos were selected for freezing. Out of these 366 thawed so far, with average survival of 74.31%. The total of the 268 thawed embryos were transferred ipsilaterally, by a non-surgical method, to 190 synchronised heifers, out of which 105 (55.26%) got in calf. Rapid freezing method based on 1.5 M of glycerol and thawing at the presence of 1.1 M sucrose proved effective and suitable for practice, as not only sufficient reviviscence of embryos and their survival in womb are guaranteed, but also a substantial shortening of the freezing as well as thawing process.     

Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI

Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors.     

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep^® Plus (AHP), Androstar^® Plus (ASP), Safecell^® Plus and TRIXcell^® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1^?/PI^?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.     

Effect of cryoprotectants and thawing temperatures on chicken sperm quality

There is need for standardization of freezing–thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post‐thaw motility and to analyse combined effect of the best permeating cryoprotectant (P‐CPA) with one of four non‐permeating cryoprotectants (N‐CPA) on post‐thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N‐methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N‐CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing–thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen‐thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post‐thaw motility. Moreover, ficoll addition to EG‐based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N‐CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.     

Combined effect of apigenin and ferulic acid on frozen‐thawed boar sperm quality

The main aim of the present study was to evaluate the cryoprotective effect of apigenin (AP) and ferulic acid (FA) on boar sperm during cryopreservation. AP and FA were both demonstrated to be high‐efficiency antioxidants and had not previously been used to protect sperm from cryodamage. As boar sperm is sensitive to oxidative stress, suitable antioxidants are still needed for improving frozen‐thawed sperm quality. With this purpose, semen samples coming from five boars were used in this study. Ejaculates of five boars were mixed and split into 16 aliquots, in which different doses of AP and FA were added separately or together. The motility, the plasma membrane integrity, the mitochondrial activity, the acrosomal integrity, the antioxidase activities and the malondialdehyde concentration of the frozen‐thawed boar sperm were assessed. The results suggested that both AP and FA significantly improved the frozen‐thawed boar sperm quality in all these aspects when they were added to the freezing extender separately, while the highest improvement was recorded when the extender was supplemented with 0.1 mmol/L AP plus 0.15 mmol/L FA. These findings demonstrated that supplementation of freezing extender with both AP and FA had a combined, beneficial effect on frozen‐thawed boar sperm.     

OPS玻璃化冷冻对小鼠四倍体胚胎体外发育的影响

为探究开放式拉长细管(OPS)玻璃化冷冻对四倍体胚胎发育的影响,本实验利用2-细胞胚胎电融合法制备四倍体胚胎,再对四倍体胚胎进行OPS玻璃化冷冻,分别观察记录二倍体胚胎、四倍体胚胎以及冷冻解冻后四倍体胚胎的发育情况。结果表明:2-细胞胚胎电融合效率为96.1%;二倍体胚胎组与电融合后四倍体胚胎组的囊胚率和孵化囊胚率差异不显著;冷冻解冻后四倍体胚胎的囊胚率(100%)与四倍体新鲜组(93.3%)差异不显著,其孵化囊胚率(72.3%)较新鲜组(64.9%)显著增高(P<0.05);四倍体冷冻解冻组的囊胚细胞数(31.96)与新鲜组(32.54)无显著差异;冷冻解冻后的四倍体早期囊胚进行体外培养时其发育速度比对照组更快。可见,冷冻对小鼠四倍体胚胎的囊胚率和囊胚细胞数均无显著影响,但孵化囊胚率显著提高,且OPS玻璃化冷冻后使四倍体胚胎的发育速度更快。     

Ultra‐Structural Alterations in In Vitro Produced Four‐Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification

Cryopreservation is the process of freezing and preserving cells and tissues at low temperatures. Controlled slow freezing and vitrification have successfully been used for cryopreservation of mammalian embryos. We investigated the effect of these two cryopreservation methods on in vitro produced four‐cell stage bovine embryos which were classified according to their quality and separated into three groups. The first group was maintained as untreated controls (n = 350). Embryos of the second (n = 385) and the third (n = 385) groups were cryopreserved either by controlled slow freezing or by vitrification. Embryos in groups 2 and 3 were thawed after 1 day. Hundred embryos were randomly selected from the control group, and 100 morphologically intact embryos from the second and third group were thawed after 1 day and cultured to observe the development up to the blastocyst stage. The blastocyst development rate was 22% in the control group, 1% in the slow‐freezing group and 3% in the vitrification group. Remaining embryos of all three groups were examined by light microscopy, transmission electron microscopy and immunofluorescence confocal microscopy with subsequent histological staining procedures. Cryopreservation caused degenerative changes at the ultra‐structural level. Compared with vitrification, slow freezing caused an increased mitochondrial degeneration, cytoplasmic vacuolization, disruption of the nuclear and plasma membrane integrity, organelle disintegration, cytoskeletal damage, a reduced thickness of the zona pellucida and a formation of fractures in the zona pellucida. Further studies are required to understand and decrease the harmful effects of cryopreservation.     

Effect of fast freezing then thaw‐aging on meat quality attributes of lamb M. longissimus lumborum

The objective of this study was to determine the effect of two different freezing rate then thaw‐aging regimens on the quality attributes of lamb loins. The loins were randomly allocated to one of five different freezing/thawing/aging regimes: fast‐(FF1A0) and slow‐(SF1A0) frozen only; fast‐(FF1A2) and slow‐(SF1A2) frozen then thaw‐aged for 14 days; aged for 14 days never frozen (A2). FF1A2 samples had a significantly higher water‐holding capacity compared to the slow frozen regardless of further aging periods. FF1A2 samples had lower (p < 0.05) shear force values than A2 and higher (p < 0.05) water‐holding capacity compared to the SF1A2. Fast freezing resulted in more intracellular cryo‐damage, whereas slow freezing resulted in extracellular cryo‐damage. FF1A0 and SF1A0 samples had lower (p < 0.05) myofibrillar proteins degradation. This study demonstrated that fast freezing then thaw‐aging can result in an improved water‐holding capacity and tenderness through the minimization of extracellular ice crystal formation, reduction in purge and drip losses, and improved proteolysis in thawed lamb.     

Cryopreservation of bovine somatic cell nuclear-transferred blastocysts: effect of developmental stage

The effect of developmental stage on the survival of bovine somatic cell nuclear-transferred blastocysts after freezing and thawing was evaluated. We also investigated how freezing affects nuclear-transferred (NT) embryos and in vitro fertilized (IVF) bovine embryos. Advanced-stage bovine NT blastocysts survived freezing better than early-stage NT blastocysts (86 vs 14%). The trend was similar with IVF embryos (87 vs 30%). At the stages tested, there was no significant difference in the survivability of NT and IVF embryos from advanced (86 vs 87%) or early-stage blastocysts (14 vs 30%). The average survival rate did not differ between NT and IVF bovine embryos (50 vs 51%). The higher survival rate of advanced-stage blastocysts compared to early-stage blastocysts in NT and IVF bovine embryos might be due to their higher cell number. In NT (128 +/- 25 vs 53 +/- 20) and IVF (128 +/- 29 vs 75 +/- 22) groups, advanced-stage blastocysts contained a significantly higher total cell number than early-stage blastocysts. There was no difference in total cell number between advanced-stage NT and IVF blastocysts (128 +/- 25 vs 128 +/- 29), however, early-stage NT and IVF blastocysts (53 +/- 20 vs 75 +/- 22) differed significantly.