The influence of cryopreservation and seminal plasma on the chromatin structure of dog spermatozoa

It was the aim of the current study to investigate effects of seminal plasma on the chromatin structure of frozen-thawed canine (Canis lupus familiaris) spermatozoa. A total of 20 ejaculates were collected. Ejaculates were divided, and one half was centrifuged for removal of seminal plasma (c) while the other was left uncentrifuged (nc) before cryopreservation. This was performed according to the Uppsala system in a computerized freezing machine. Before freezing (bf) and after thawing (at), samples were investigated for motility (M), viability (CASA), and chromatin status (sperm chromatin structure assay; SCSA). Before freezing, the average DFI% and the SD-DFI from 20 nc ejaculates were 1.7 ± 4.0% and 18.6 ± 1.2, respectively. After thawing, all motility parameters decreased and were significantly lower in centrifuged than in noncentrifuged samples, whereas the percentage of morphologically abnormal spermatozoa (Morph) was significantly higher (nc: M bf, 84.1 ± 20.6%; M at, 51.9 ± 15%; c: M bf, 84.1 ± 20.6%; M at, 43.3 ± 22.2%; Morph nc: 28.3 ± 7.8% vs. c: 31.0 ± 9.8%). Furthermore, only in c samples did the DFI increase within 6 h after thawing (DFI c: bf, 41.8 ± 1.5%; 6 h at, 45.4 ± 6.6%; P < 0.01). The SD-DFI as well as the DFI% increased within 3 h of storage in both groups (SD-DFI nc: bf, 18.6 ± 1.2%; 3 h at, 25.8 ± 5.4%; DFI% nc: bf, 1.1 ± 4.0%; 3 h at, 6.1 ± 12.9%; P < 0.05). For both parameters, there was no significant difference between c and nc samples at any time investigated. In conclusion, centrifugation of semen samples before freezing decreased postthaw motility and increased the percentage of morphologically abnormal spermatozoa as well as the degree of sperm chromatin denaturation over time. Centrifugation of canine ejaculates before cryopreservation can therefore no longer be recommended.     

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved (P < 0.05) by the addition of 10 μM Y-27632 (94.9 ± 2.4%, mean ± SEM) compared to a control (78.0 ± 6.0%). Conversely, after 48 h of culture, there were no significant differences in hatching rate (62.8 ± 11.1 vs. 59.6 ± 9.4%) and mean total cell number (135.2 ± 13.1 vs. 146.7 ± 13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7 ± 3.8 vs. 54.7 ± 8.9%, P < 0.05), with no difference in mean total cell number of blastocysts (230.0 ± 23.0 vs. 191.2 ± 22.2, P = 0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5 ± 2.1, 31.4 ± 2.3, 36.2 ± 3.2, and 28.6 ± 6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.     

The effect of insemination time and sperm dose on pregnancy rate using sex-sorted ram sperm

The objective was to determine the optimum timing of insemination and minimum effective dose rate of sex-sorted ram sperm. Semen from three Merino rams was sorted into high purity X- and Y-chromosome bearing sperm populations. Ovulation was controlled in 732 Merino ewes using PMSG at progestagen pessary removal and GnRH 36 h later. Sorted (S) and non-sorted (NS) doses of 1 or 15 × 10⁶ motile, frozen-thawed sperm were inseminated laparoscopically at 50, 54, 58, 62, and 66 h after progestagen withdrawal. An additional treatment dose of 0.5 × 10⁶ S or NS sperm was inseminated at the 58 h time point (n = 60). Pregnancy was diagnosed by ultrasound at 60-62 d gestation. Both 1 × 10⁶ and 15 × 10⁶ sperm achieved similar pregnancy rates, regardless of sperm type, at 58 h (S1: 46 ± 9.4%; S15: 43 ± 9.3%; NS1: 41 ± 9.2%; NS15: 49 ± 9.4%). However, pregnancy rates were lower (P < 0.05) for doses of 1 than 15 × 10⁶ sperm inseminated at 50 (15 ± 6.3% vs. 36 ± 9.1%), 54 (14 ± 4.4% vs. 55 ± 7.3%), 62 (33 ± 6.9% vs. 54 ± 7.3%), and 66 h (29 ± 8.6% vs. 56 ± 9.5%). There was no difference between S and NS sperm for inseminations with 0.5 × 10⁶ motile sperm at 58 h after PR (15 ± 3.6% vs. 14 ± 3.3%), nor with 15 × 10⁶ motile sperm at all insemination times (49 ± 6.3% vs. 49 ± 6.3%). However, fertility was higher for S than NS sperm at the 1 × 10⁶ dose level (37 ± 6.1% and 16 ± 4.0%). More than 90% of lambs born were of the predicted sex. We hypothesise that the sorting process selects a homogeneous, fertile sub-population of sperm, removing those that are dead, damaged and morphologically abnormal.     

Developmental competence and mRNA expression of preimplantation in vitro-produced embryos from prepubertal and postpubertal cattle and their relationship with apoptosis after intraovarian administration of IGF-1

Recombinant human Insulin-like growth factor-I (hIGF-1) was administered to one ovary of prepubertal and postpubertal cattle to determine its effects on (1) oocyte developmental competence, (2) the expression pattern of six developmentally important genes (GLUT3, GLUT8, AKT1, BCL-XL, BAD, and BAX), and (3) its relationship with apoptosis (female Holstein-Friesian). Oocytes were retrieved from 7- to 10-mo-old prepubertal dairy calves (preP), 11- to 18-mo-old postpubertal heifers (postP), and cows via ultrasound-guided follicular aspiration. Immature oocytes were matured in vitro then fertilized and cultured up to the blastocyst stage. Apoptosis was determined by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) in 8-d blastocysts. Similar low blastocyst yields were observed in the IGF-1-treated preP group (11.2 ± 2.4%), the control preP group (10.4 ± 3.0%), and in the IGF-1 postP group (10.9 ± 2.3%). These were lower (P ≤ 0.01) compared with the control postP group (21.2 ± 3.8%) and with cows (23 ± 3.7%). The expression profile of the six genes was partly affected by age and IGF-1 treatment. Apoptosis was correlated with the age of the oocyte donors and was increased in blastocysts derived from prepubertal heifers. Results show that apoptosis is a critical feature of the acquisition of developmental competence of oocytes from prepubertal cattle and that IGF-1 did not beneficially affect oocyte developmental competence.     

Improvement in buffalo (Bubalus bubalis) spermatozoa functional parameters and fertility in vitro: Effect of insulin-like growth factor-I

The aim of the current study was to assess the effect of insulin-like growth factor-I (IGF-I; 100 ng/mL) on buffalo (Bubalus Bubalis) sperm functional parameters related to in vitro fertilization. The acrosin activity (the mean diameter of halo formation in micrometers) was significantly higher in the IGF-I group (14.17 ± 1.51) compared with that in the control group (9.50 ± 0.36) at 2 h incubation. The mitochondrial membrane potential (per cent) was significantly higher in the IGF-I group compared with that in the control group at 30 min (33.27 ± 2.62 vs. 26.71 ± 1.02), 60 min (24.24 ± 3.45 vs. 18.77 ± 2.09), and 90 min (22.86 ± 3.02 vs. 16.92 ± 1.24) incubation. The percentage of spermatozoa positive for sperm nuclear chromatin decondensation (NCD) differed significantly between the groups at 90 and 120 min incubation. The comet length was significantly lower in the IGF-I group compared with that in the control group at 2 h incubation. The percentage of fragmented DNA in the tail did not differ significantly between the groups at 2 h incubation. The percentage of acrosomal-reacted spermatozoa did not differ significantly between the IGF-I and the control groups at 4 h (41.12 ± 6.44 vs. 43.53 ± 5.05) incubation. The cleavage rate (per cent) was significantly higher in the IGF-I-treated group (56.73 ± 3.70) compared with that in the control group (44.85 ± 2.15). The current study suggests that the addition of IGF-I prevents deterioration of sperm functional parameters and fertility.     

Effect of droplet vitrification on development competence, actin cytoskeletal integrity and gene expression in in vitro cultured mouse embryos

The effect of modified droplet vitrification was assessed on cellular actin filament organization, apoptosis related gene expression and development competence in mouse embryos cultured in vitro. Mouse zygotes, 2-cell embryos and morulae were vitrified in ethylene glycol (VS-1) and ethylene glycol plus DMSO (VS-2) and thawed by directly placing the vitrified drop into 0.3 M sucrose solution at 37 °C. High recovery (93-99%) of morphologically normal embryos was evident following vitrification and thawing. No detectable actin filament disruption was observed in the embryos at any development stage following vitrification and thawing and/or in vitro culture. The expression pattern of Bax, Bcl2 and p53 genes was altered (P < 0.05) in vitrified zygotes and 2-cell embryos, but not in morulae. Although a large proportion of the vitrified zygotes (59.5 ± 4.4% in VS-1 and 57.9 ± 4.5% in VS-2; mean ± S.E.M.) and 2-cell embryos (63.1 ± 4.4% in VS-1 and 59.2 ± 4.3% in VS-2) developed into blastocysts, development of control embryos (70.2 ± 5.0% of zygotes and 75.5 ± 4.4% of 2-cell embryos) into blastocysts was higher (P < 0.05). In contrast, development of the control and vitrified morulae into blastocysts (more than 85%) was similar. We concluded that the modified droplet vitrification procedure supported better survival of morula stage compared to zygotes and 2-cell mouse embryos.     

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142 ± 21.8 vs. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P < 0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P < 0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading.     

Effect of storage of domestic cat (Felis catus) epididymides at 5 °C on sperm quality and cryopreservation

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91 ± 2.02, 48.21 ± 1.47, and 43.03 ± 1.32) and after thawing (51.81 ± 3.02, 41.90 ± 2.14, and 42.35 ± 1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33 ± 1.38% before and 52.50 ± 1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22 ± 1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI = 39.17 ± 2.76 and 45.00 ± 2.65, respectively) and the percentage of intact acrosomes (45.76 ± 4.91% and 60.67 ± 3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24 h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.     

Vitrification and warming of in vivo-derived porcine embryos in a chemically defined medium

The aim of this study was to design a protocol for vitrification and warming of porcine embryos in a chemically defined medium. A total of 663 morulae and blastocysts were collected from weaned crossbred sows (Large White-Landrace) 5 to 6 d after estrus and vitrified with the Superfine Open Pulled Straw method. In Experiment 1, embryos were vitrified using as a basic medium TCM-199-HEPES supplemented with 20% newborn calf serum (NBCS) or with 0, 0.1%, 0.5%, or 1% polyvinyl alcohol (PVA). Nonvitrified embryos were used as a fresh control group. Survival and hatching rates were evaluated after 72 h of in vitro culture to assess embryo viability. In addition, some hatched blastocysts derived from morulae and blastocysts were processed to determine the total cell number and the cell proliferating index as measures of their quality. Within each stage of embryo development, the different vitrification groups and the fresh control group showed similar high embryo survival (range, 70.5 ± 7.1% to 84.9 ± 8.1% and 85.3 ± 8.1% to 98.4 ± 8.2% for morulae and blastocysts, respectively) and hatching rate (range, 46.3 ± 10.1% to 66.7 ± 11.2% and 73.7 ± 11.3% to 89.4 ± 11.2% for morulae and blastocysts, respectively) and quality after in vitro culture. In Experiment 2, embryos were vitrified using 0.1% PVA and warmed with TCM-199-HEPES-0.13 M sucrose supplemented with 20% NBCS or either 0 or 0.1% PVA. Nonvitrified embryos were used as a fresh control group. As in Experiment 1, no significant differences were detected in embryo survival (range, 67.9 ± 6.6% to 74.5 ± 6.6% and 91.9 ± 7.0% to 99.5 ± 6.3% for morulae and blastocysts, respectively) and hatching rate (range, 47.0 ± 7.2% to 64.8 ± 9.9% and 89.4 ± 7.4% to 98.2 ± 6.9% for morulae and blastocysts, respectively) and quality among the warming groups or among vitrified and fresh control embryos. In both experiments, the developmental embryo stage influenced the survival and hatching rates, as well as the number of cells (P < 0.01), with the blastocyst stage yielding the best results. In conclusion, PVA can be used as a substitute for serum in vitrification and warming solutions without detrimental effects on the in vitro development of in vivo-derived porcine morulae and blastocysts.     

Embryo production after in vitro fertilization with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm

The objective of this study was to determine the in vitro fertilizing capacity of bull sperm derived from fresh or frozen samples and subjected to sex sorting and re-cryopreservation. Four sperm types were assessed for their ability to fertilize and sustain early embryo development in vitro. Semen from three Bos taurus bulls of different breeds (Jersey, Holstein and Simmental) was collected and either sorted immediately and then frozen (SF) or frozen for later sorting. Frozen sperm destined for sorting were thawed, sex-sorted, and re-frozen (FSF) or thawed, sex-sorted (FS), and used immediately for in vitro fertilization (IVF). Frozen-thawed nonsorted semen from the same ejaculate was used as a control. Oocytes from donor cows were aspirated via ovum pick-up and matured in vitro prior to IVF and culture. On average, 19.0 ± 1.7 (mean ± SEM) oocytes were aspirated per donor cow, of which 74.4 ± 2.2% were selected for maturation. The proportion of cleaved embryos (Day 3) did not differ between sperm groups (P = 0.91). Likewise, IVF with FSF sperm resulted in similar Day 7 blastocyst rates (as a percentage of total oocytes) as those of control, SF, and FS sperm (FSF, 34.5 ± 4.7; control, 32.2 ± 4.6; SF, 35.9 ± 4.8; and FS, 26.9 ± 4.1%; P = 0.23). These encouraging results show that frozen-thawed sex-sorted sperm may be re-frozen and used for in vitro embryo production with similar blastocyst production as that of nonsorted frozen-thawed and sex-sorted frozen-thawed sperm.     

Apoptosis in fresh and cryopreserved buffalo sperm

The objectives of present study were (a) validation of annexin V/PI assay for estimation of sperm apoptosis in buffalo (Experiment 1) and (b) determining the effect of stages of cryopreservation on sperm apoptosis and its correlation with sperm motility and plasma membrane integrity (Experiment 2). In Experiment 1, different levels of apoptosis were artificially induced in buffalo semen (100 × 10⁶ sperm/aliquot) through graded doses of camptothecin (5, 10 and 20 μM/aliquot). Higher concentrations of camptothecin (10 and 20 μM) successfully (P < 0.05) induced apoptosis as compared to the lower (5 μM) dose and/or control. In Experiment 2, semen samples (n = 9, three pooled semen samples from each of the three buffalo bulls separately) were cryopreserved using vapor freezing. The mean percentage of apoptotic, necrotic and viable sperm did not differ between fresh and before freezing stages. However, freezing and thawing increased (P < 0.05) the percentage of apoptotic sperm (25.4 ± 0.6 vs. 36.5 ± 1.9) while decreased (P < 0.05) the necrotic (35.1 ± 1.2 vs. 29.7 ± 0.7) and viable sperm (37.2 ± 1.3 vs. 32.8 ± 1.9, (P < 0.07). Likewise, the mean percent motility and plasma membrane integrity decreased (P < 0.05) (64 ± 2.1 vs. 49.4 ± 1.3) and (79.6 ± 0.5 vs. 38.7 ± 0.3) respectively, at post thaw compared to other stages. Coefficient of correlation, combined at all stages for each variable revealed that sperm apoptosis was inversely correlated with sperm motility and plasma membrane integrity. It is concluded that (a) the annexin V/PI assay can be used as a tool to determine the buffalo semen apoptosis and (b) freezing and thawing induces apoptosis in buffalo sperm.     

Effects of bovine Herpesvirus Type 5 on development of in vitro-produced bovine embryos

Bovine (Bos indicus) herpesviruses have been associated with reproductive disease. Type 1, the most studied species, is best known for its reproductive and respiratory effects. Type 5 (BoHV-5) has been detected in bull semen and aborted fetuses but not in oocytes and embryos. This study consisted of three experiments that evaluated (1) BoHV-5-infected oocytes matured in medium with fetal bovine serum (BoHV-FBS) or polyvinyl alcohol (BoHV-PVA) and fertilized by noninfected sperm; (2) noninfected oocytes fertilized by BoHV-5-infected sperm; and (3) infection of presumptive zygotes by BoHV-5. Each treatment involved nine drops of 15 to 20 oocytes. Infection with BoHV-5 was detected by polymerase chain reaction and in situ hybridization assay, and fertilization capacity and embryonic development were assessed using in vitro culture. Experimentally induced infection was obtained in all experiments, and vertical transmission of BoHV-5 by gametes was confirmed. The cleavage rate was reduced (P = 0.0201) in BoHV-FBS (80.4 ± 8.9%; mean ± SD) compared with that of noninfected oocytes (89.9 ± 6.5%); neither differed from BoHV-PVA (87.3 ± 7.1%), and the resulting embryo production rate was not significantly different among groups. Rates of cleavage (87.5 ± 7.5% vs. 92.2 ± 5.5%, control vs. infected) and development of embryos (41.7 ± 9.9% vs. 44.3 ± 7.7% to morula/blastocyst/expanded blastocyst [M/B/EB] and 39.6 ± 10.3% vs. 40.8 ± 9.2% to blastocyst/expanded blastocyst/hatching blastocyst [B/EB/HB] stages) were not compromised by infected sperm (P = 0.1462, P = 0.5402, and P = 0.8074, respectively). However, presumptive zygotes directly infected 1 d after fertilization produced a lower number (P = 0.0140 to M/B/EB and P = 0.002 to B/EB/HB stages) of in vitro-produced embryos (31.6 ± 4.6 vs. 25.0 ± 5.5 and 31.6 ± 4.6 vs. 20.2 ± 5.4; control vs. infected). In conclusion, BoHV-5 infected gametes and was transmissible to the embryo during in vitro development. As zygotes infected 1 d after fertilization had compromised development, BoHV-5 has the potential to be a pathogen with economic consequences.     

Effect of semen quality in transgenic boars on the developmental competence of preimplantation embryos

The aim of this study was to compare the fertilising capacity of sperm from 6 transgenic (TG) and 6 non-transgenic (NTG) boars based on analyses of embryos resulting from insemination with sperm from these particular boars. Expanded blastocysts were collected from five groups of synchronised gilts (six gilts per group) inseminated by TG boars bearing a gene construct containing the human α1,2-fucosyltransferase gene and by NTG boars. The ejaculates used for insemination were analysed to detect apoptotic changes using two fluorescence methods: an assay to assess early changes in the membrane integrity of the sperm using the YO-PRO-1 fluorophore and an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labelled Annexin-V. Our results, using a combination of YO-PRO-1 and PI fluorophores, revealed no significant differences in the percentage of sperm subpopulations between non-transgenic and transgenic boars (P < 0.01). Moreover, the second fluorescent probe also revealed no significant differences between the average values of live (Ann-V⁻/PI⁻), early apoptotic (Ann-V⁺/PI⁻), and late apoptotic/early necrotic sperm (Ann-V⁺/PI⁺) as calculated for TG and NTG boars. Only the percentage of necrotic sperm (Ann-V⁻/PI⁺) was significantly different (P < 0.05) between transgenic and non-transgenic boars (3.4% ± 2.7; 7.2% ± 2.1, respectively). The quality of the preimplantation embryos at the blastocyst stage was determined by counting the number of cells, observing a TUNEL-positive reaction and by caspase-3 labelling. We found that expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen showed almost no DNA fragmentation (80%) and 70% caspase-3 activity. The expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen did not differ significantly in their DNA fragmentation, and there were no differences in caspase-3 activity. These results revealed a positive correlation between the percentage of blastocysts with TUNEL-positive nuclei and the percentage of blastocysts with caspase-3 activity (r = 0.9787; P < 0.0001).     

Cryobanking as tool for conservation of biodiversity: effect of brown trout sperm cryopreservation on the male genetic potential

Sperm cryobanking could be a good alternative to breeding in captivity in order to preserve genetic diversity. Sperm from two well-characterized brown trout populations originating from two river basins in the Northwest of Spain (Esla and Duerna), both threatened by extinction, was cryopreserved. In order to determine whether a sperm cryobank is the best option for preserving genetic profiles, cell viability, chromatin fragmentation, fertility and genetic variability of the offspring obtained with fresh and frozen sperm, were analyzed. Sperm viability was not reduced by freezing (87.0 ± 3.32% to 77.9 ± 3.59% and 77.6 ± 6.53% to 76.6 ± 2.61% in fresh and frozen sperm from Esla and Duerna, respectively). The percentage of fragmented DNA increased after freezing in spermatozoa from Esla males (from 4.7 ± 0.23% to 6.0 ± 0.28%), but not those from Duerna males.After freezing/thawing, the percentage of eyed embryos drops from 66.8 ± 6.77% to 16.1 ± 3.46% and from 50 ± 8.97% to 11.5 ± 2.50% in the Esla and Duerna basins, respectively. This reduction indicates that many spermatozoa have lost their ability to contribute to embryo development and this loss is not related to either spermatozoa viability or the DNA integrity. Genotypic determination by microsatellite analysis showed that frozen/thawed sperm provided offspring with a similar genetic profile to unfrozen milt, demonstrating the accuracy of the cryopreservation procedure.Taking into account the prolificacy of this species, even a low rate of success of fry after cryopreservation, could provide enough individuals to recover stable populations without altering the genetic profiles of the preserved strains. Therefore, cryopreservation is considered a safe, simple and cheap technology for gene banking in the analyzed brown trout populations.     

Effect of sperm treatment on efficiency of EGFP-expressing porcine embryos produced by ICSI-SMGT

Intracytoplasmic sperm injection-sperm-mediated gene transfer (ICSI-SMGT) is a useful tool for the production of transgenic mice but is still rather inefficient in farm animals. In the current study, we evaluated the effect of the sperm treatments on the efficiency for producing enhanced green fluorescent protein (EGFP)-expressing pig embryos by ICSI-SMGT. Four different sperm treatments were assayed: (1) fresh (control), (2) frozen-thawing (FT), (3) quick freezing without cryoprotectant agents (QF), and (4) Triton X-100 treatment (TX-100). First, we evaluated the DNA-binding ability and the viability of sperm under the different treatments coincubated with exogenous DNA (EGFP) by flow cytometry. Second, we evaluated the embryo production rate and the efficiency in transgene expression in embryos after using these spermatozoa to fertilize oocytes by ICSI. Sperm treatment significantly increased DNA-binding capacity but reduced sperm viability compared with that of the control group. Treatments damaging the spermatozoa's membranes (QF and TX-100) resulted in a greater capacity of sperm binding exogenous DNA than that after FT treatment (P < 0.01). Similar rates of EGFP-expressing embryos were obtained from the control, FT, and TX-100 groups (37.04 ± 3.52%, 43.54 ± 5.41%, and 29.03 ± 8.29%, respectively), but were significantly higher in the QF group (80.43 ± 5.91%). These results demonstrate that the integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions between an injected exogenous DNA and the sperm chromatin. However, severe sperm treatments such as QF and TX-100 may damage the sperm nucleus, induce DNA fragmentation, and/or lead to chromosomal breakage with a detrimental effect on further embryonic development.     

Comparison of two different cryopreservation protocols for freezing goat semen

In this study, two different semen cryopreservation protocols were compared to freeze goat semen. The ejaculates (n = 12) were collected by using electro-ejaculator from six mature bucks (two ejaculates per each buck). Each ejaculate was divided into two groups as Protocol 1 (P1) and Protocol 2 (P2). In P1, semen was diluted directly in an extender containing 15% egg yolk, 300 mM Tris, 28 mM glucose, 95 mM citric acid 5% glycerol to a concentration of 200 × 10⁶ sperm/mL. In P2, after the removal of seminal plasma by centrifugation, the semen sample was diluted with the first portion of milk extender consist of 100 mg/mL skimmed milk powder and 27.75 mM glucose (without glycerol) to a concentration of 400 × 10⁶ sperm/mL. The second portion of the milk extender containing 14% glycerol was added to semen gradually in order to achieve sperm concentration 200 × 10⁶ sperm/mL and 7% glycerol level in the final volume. Extended semen was loaded in 0.25 mL straws, held for 2 h at 4 °C, frozen in nitrogen vapor and stored in liquid nitrogen. Post-thaw motility and live sperm rate (mean ± SEM) were significantly lower (P < 0.05) in P1 as compared to P2 (47.50 ± 1.23% vs. 55.63 ± 1.72%; 80.04 ± 1.29% vs. 84.04 ± 1.08%, respectively). However, live intact, total intact, abnormal, reacted acrosome and DNA damaged sperm rates were similar (P > 0.05) in both protocols. It was concluded that both protocols used in this study provided reasonable post-thaw parameters; however, P2 yielded better motility and live sperm rate compared to P1.     

Sperm quality and the morphology of cryopreserved testicular tissues recovered post-mortem from diverse wild species

This study compared the effects of slow and fast freezing of testicular tissue of wild animals collected at post-mortem on testicular structure and testicular sperm. The testes of seven animals that had died in captivity; three felids (jungle cat, lion and leopard), two cervids (rusa deer and fea’s muntjac) and two bovids (Sumatran serows) were cryopreserved using slow- and fast-freezing protocols. There were greater reductions in the integrity of the sperm membrane and DNA in tissues cryopreserved using slow freezing compared to fast freezing (membrane integrity reduced by 21.5 ± 12.4% vs. 13.0 ± 6.9%, P = 0.11 and DNA integrity reduced by 22.7 ± 16.3% vs. 6.6 ± 6.3%, P = 0.13). Histologically, there were similar degrees of detachment and shrinkage of the seminiferous tubules whereas, TUNEL assay revealed a tendency towards more apoptotic changes in the intra-tubular cells of tissues frozen using fast freezing compared to slow freezing (P = 0.09). In conclusion, fast freezing tended to cause less damage to testicular sperm but its protective effect on intra-tubular cells was likely compromised. This is the first report of gamete recovery in the wild and of the comparison in various wildlife species, between testicular tissues cryopreserved using different protocols.     

Enhanced histone acetylation in somatic cells induced by a histone deacetylase inhibitor improved inter-generic cloned leopard cat blastocysts

The objective was to determine whether alterations of histone acetylation status in donor cells affected inter-generic SCNT (igSCNT)-cloned embryo development. Leopard cat cells were treated with trichostatin A (TSA; a histone deacetylase inhibitor) for 48 h, and then donor cells were transferred into enucleated oocytes from domestic cats. Compared to non-treated cells, the acetylated histone 3 at lysine 9 (AcH3K9) and histone 4 at lysine 5 (AcH4K5) in the TSA group increased for up to 48 h (P < 0.05). The AcH3K9 signal ratios of igSCNT group was higher than control group 3 h after activation (P < 0.05). Treatment with TSA significantly increased total cell number of blastocysts (109.1 ± 6.9 vs. 71.8 ± 2.9, mean ± SEM), with no significant effects on rates of cleavage or blastocyst development (71.1 ± 2.8 vs. 67.6 ± 2.9 and 12.2 ± 2.6 vs. 11.0 ± 2.6, respectively). When igSCNT cloned embryos were transferred into a domestic cat oviduct and recovered after 8 d, blastocyst development rates and total cell numbers were greater in the TSA-igSCNT group (20.7 ± 3.0% and 2847.6 ± 37.2) than in the control igSCNT group (5.7 ± 2.2% and 652.1 ± 17.6, P < 0.05). Average total cell numbers of blastocysts were approximately 4.4-fold higher in the TSA-igSCNT group (2847.6 ± 37.2, n = 10) than in the control group (652.1 ± 17.6, n = 8; P < 0.05), but were ∼2.9-fold lower than in vivo cat blastocysts produced by intrauterine insemination (8203.8 ± 29.6, n = 5; P < 0.001). Enhanced histone acetylation levels of donor cells improved in vivo developmental competence and quality of inter-generic cloned embryos; however, fewer cells in blastocysts derived from igSCNT than blastocysts produced by insemination may reduce development potential following intergeneric cloning (none of the cloned embryos were maintained to term).     

Semen characteristics of genetically identical male cats cloned via somatic cell nucleus transfer

We investigated the sperm characteristics of four cloned male cats (Felis catus) to assess their reproductive potential. Fresh and frozen-thawed sperm were assessed for motility, viability, and morphology, and their functional competence was evaluated by in vitro fertilization (IVF) of domestic cat oocytes. All fresh semen characteristics varied among cats and collection times. Sperm concentration (× 10⁶/mL) of Cat A (512 ± 140, range 368 to 685) was significantly higher, whereas that of Cat C (335 ± 92, range 274 to 469) was significantly lower than that of Cloned B (459 ± 159, range 336 to 510) and control cats (680 ± 452, range 360 to 479). After thawing, motility and progressive motility of sperm from Cat B were significantly lower than that of the other cloned and control cats. The curvilinear, straight line, and average path velocities of sperm from Cat B were significantly higher, whereas the straightness was lower, than that of the other cloned and control cats. Frozen sperm from Cats A, B, and C successfully fertilized oocytes (cleavage = 74.4%, 71.4%, and 86.2%, respectively) and produced embryos that developed to the blastocyst stage after IVF/In vitro culture (IVC) (34.4%, 26.7%, and 48.0%) at frequencies similar to the cleavage rate (82.0%) and blastocyst rate (43.9%) obtained with sperm from the control male. In conclusion, seminal characteristics of cloned male cats did not differ markedly from those of our noncloned, control male cats.     

Single-layer centrifugation through colloid selects improved quality of epididymal cat sperm

The objectives were to determine the: 1) extent of epithelial and red blood cell contamination in epididymal cat sperm samples recovered by the cutting method; 2) efficacy of simple washing, single-layer centrifugation (SLC), and swim-up for selecting epididymal cat sperm; and 3) effects of freezing and thawing on cat sperm selected by various techniques. Ten unit samples were studied; each contained sperm from the cauda epididymides of four cats (total, ∼200 × 10⁶ sperm) and was equally allocated into four treatments: 1) simple washing, 2) single-layer centrifugation through colloid prior to cryopreservation (SLC-PC), 3) single-layer centrifugation through colloid after cryopreservation (SLC-AC), and 4) swim-up. Centrifugation (300 × g for 20 min) was done for all methods. The SLC-PC had a better recovery rate than the SLC-AC and swim-up methods (mean ± SD of 16.4 ± 8.7, 10.7 ± 8.9, and 2.3 ± 1.7%, respectively; P < 0.05). The SLC-PC, SLC-AC and swim-up samples contained less red blood cell contamination than simple washed samples (0.02 ± 0.01, 0.02 ± 0.04, 0.03 ± 0.04, and 0.44 ± 0.22 × 10⁶ cells/mL, respectively; P < 0.05). Although the proportion of sperm with head abnormalities did not differ among selection methods (P > 0.05), SLC-PC yielded the highest percentage of sperm with normal midpieces and tails (P < 0.05), due to the lowest proportion of coiled tails (P < 0.05). Furthermore, the SLC-PC was as effective as swim-up in removing sperm with proximal droplets, and selecting motile sperm, as well as those with intact membranes and DNA (P > 0.05). In conclusion, both SLC-PC and swim-up improved the quality of epididymal cat sperm, including better morphology, membrane and DNA integrity, and removal of cellular contamination. However, SLC had a better sperm recovery rate than swim-up.