Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: Efficiency and in vitro developmental competence

The production of embryos with a preselected sex sperm is important in the livestock industry. In this study, we examined the efficiency of producing female embryos by intracytoplasmic sperm injection (ICSI) with flow cytometry sorted (ssICSI) and unsorted (usICSI) bovine sperm, and their developmental competence in vitro. For comparison, bovine embryos were also produced by in vitro fertilization (IVF) with sorted (ssIVF) and unsorted (usIVF) bovine sperm. The semen used in this study was from a bull selected for its high fertility and blastocyst developmental competence among four bulls. We first examined and compared pronuclear (PN) formation and cleavage rates of the produced embryos among the treatment groups. Our results demonstrated that PN formation rates (judged by two pronucleus [2PN]) and cleavage rates in ssIVF group (23.1% and 43.6%) were lower than those in the usIVF (71.1% and 71.6%), usICSI (73.1% and 92.8%) and ssICSI (75% and 79.1%) groups, respectively (P < 0.05). Moreover, the blastocyst formation rate in the ssIVF group was less than those in the usIVF, usICSI, and ssICSI groups (2.7% vs. 30.2%, 28.7% and 24.7%, respectively; P < 0.05). Importantly, we reported that the blastocyst formation rate in the ssICSI group was similar to that in the usICSI group, which indicated that ICSI can rescue the damage introduced to sperm by flow cytometry–mediated sex-sorting. Of note, we achieved a blastocyst formation rate in the ssICSI group to be comparable with the usIVF group. We then examined embryo quality by counting the number of normal and apoptotic cells in blastocysts. It was found that, despite the fact that blastocyst formation rate in the ssIVF group was significantly lower than those in the usIVF, usICSI and ssICSI groups, there was no difference in total and apoptotic cell numbers among these groups (P > 0.05). Finally, karyotyping analysis demonstrated that the proportion of female embryos in the ssICSI and ssIVF groups was 100%, whereas it was 58.8% and 57.8% in the usIVF and usICSI groups, respectively. In conclusion, ICSI with flow cytometry sorted bovine sperm provides an alternative approach to produce embryos with predetermined sex.     

Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30–36-month-old, 500–550 kg) with good seminal quality (>80% motile and morphologically normal sperm) had scrotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1–5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P < 0.05) 14 and 21 d after insulation was removed. On 14 and 21 d, the incidence of head defects (9.7 ± 0.6 and 17.0 ± 0.8, respectively; mean ± S.E.M.) was higher (P < 0.05) in agreement with the incidence of nuclear vacuoles (14.0 ± 5.0 and 12.3 ± 2.3) and abnormal chromatin (24.4 ± 7.2 and 30.8 ± 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P < 0.05), blastocyst rates were lower (P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R² = 0.38, 0.48, and 0.30, respectively) and blastocyst rates (R² = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R² = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm.     

Maternal age effect on early human embryonic development and blastocyst formation

In humans, age-related decline in female fertility can be explained by a reduction in quality either of the older uterus or of the embryos arising from aging oocytes. The aim of this study was to examine the latter hypothesis, using in vitro fertilization (I.V.F.) and coculture of embryos until the blastocyst stage. We determined the blastocyst formation rate ([expanded blastocysts/blastocysts]*100) according to the patient's age the day of I.V.F. With increase in age, the number of retrieved oocytes decreased, without alteration of the cleavage rate. In patients above age 30 years, preimplantation development to blastocysts declined due to an increase in embryo arrest at the morula stage. If blastocyst stage was reached, a negative linear relationship between blastocyst expansion rate and patient age was observed. Drops in gamete production and embryo development with increasing age led to a drastic decrease in patients having at least one expanded blastocyst (<30 years, 82%; ≥40 years, 36%). A high delivery rate per oocyte retrieval (25.8%) was observed in patients above age 40 years after embryo transfer at the blastocyst stage. These results give a clear indication of decline in the quality of human embryos arising from aging oocytes. The origin of this alteration is discussed in terms of chromosome abnormalities, role of maternally-inherited products from the oocyte, timing of genomic activation, and temporal pattern of gene expression during initial development of the human embryo. © 1996 Wiley-Liss, Inc.     

Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22–24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me₂SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 μM 2′7′ dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV⁻ PI⁻), early apoptotic (AV⁺ PI⁻), dead non-apoptotic (AV⁻ PI⁺) and necrotic (AV⁺ PI⁺). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 μM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.     

The retention of 125I-labeled plasma membrane proteins in bovine spermatozoa cryopreserved in egg-yolk citrate extender

Cryopreservation of bovine sperm in egg-yolk citrate extender (EYC) usually maintains fertility. Since plasma membrane proteins are important for the fertilizing potential of sperm, the possible loss of membrane proteins from sperm subjected to cryopreservation in EYC was evaluated. Sperm were washed and labeled with ¹²⁵I without significantly reducing motility. Radiolabeled sperm were a) held for 2 hr at 22°C in N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES)-buffered saline containing 1% polyvinyl alcohol, b) cooled to 5 °C in glycerol-free EYC and held for 3 hr, or c) frozen-thawed in EYC containing 7% glycerol. Sperm were solubilized and proteins were separated by electrophoresis under denaturing conditions. Freeze-thawing dislodged most egg-yolk proteins from the spermatozoal plasma membrane that were bound to and retained by sperm that only were cooled to 5 °C. Autoradiography resolved 11-18 bands of ¹²⁵I polypeptides. There was no difference (P > 0.05) in the amount of ¹²⁵I protein retained by frozenthawed and cooled sperm. However, the radioactivity in two polypeptide bands (MW = 105 K and 24.2 K) was less (P < 0.05) for sperm held at 22 °C in HEPES-buffered saline. Thus, holding sperm in buffered saline at 22 °C resulted in a greater loss of ¹²⁵I proteins from the plasma membrane than did cryopreservation of sperm in EYC. Cryopreservation did not induce greater loss of ¹²⁵I proteins from the plasma membrane than simply cooling sperm to 5 °C in EYC.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

Effect of overdose zinc on mouse testis and its relation with sperm count and motility

The purpose of this study was to investigate the effects of excessive zinc intake on the testes and on sperm count and motility in mice. Thirty Balb c mice were divided randomly into 3 groups of 10 animals in each. Group I acted as controls; group II was supplied with drinking water containing 1.5 g/100 mL Zn, and group III was supplied with drinking water containing 2.5 g/100 mL Zn. The animals were sacrificed after 3 wk supplementation and the epididymis and testis were quickly excised. A negative correlation between Zn dose and sperm count and motility was found. The sperm count in group III was significantly lower than in groups II and I (p<0.05). The sperm motility in group III was significantly lower than in the controls (p<0.05). Degenerative changes, including spermatic arrest, degeneration of seminiferous tubules, and fibrosis in interstitial tissue, were observed in group III animals. These results show that high doses of zinc significantly alter sperm motility.     

Animal Posters

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P?<?0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P?<?0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P?>?0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P?>?0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P?>?0.05) in the presence of heparin and PHE (P?<?0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (47.5% vs. 71.6%, p<0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p>0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36 to 42 h (83.6% vs. 96.7%, p<0.05). However, no statistical difference was observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p>0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O₂) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O₂) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.3%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.     

Comparison of sperm viability with seminal plasma metal levels

This study compares the semen levels of lead (Pb), cadmium (Cd), and aluminum (Al) in relation to live sperm in semen samples from 64 apparently healthy men. The measured levels were separated into live sperm count tertiles (<25% [18 subjects], 25–50% [26 subjects], and >50% [20 subjects]). The mean ± SD for each group was calculated, and the difference between the means of the high and low tertiles were compared by ANOVA. Significant differences were observed between the high and low live sperm groups for Pb (p < 0.01) and Al (p < 0.05), but not Cd. Spearman’s rank correlation between sperm viability and the semen plasma metal levels showed a direct relation to Mg (p < 0.05). However, there was an inverse relation to lead (p < 0.001), cadmium (p < 0.01), and aluminum (p < 0.01). There was no significant correlation between Ca and Zn. Linear regression between the live sperm counts and semen level of the three metals show that metal levels were inversely correlated with the percentage of live sperm (p < 0.001, <0.01). Apparently, the presence of these metals in the environment and in seminal plasma exerts a toxic effect on sperm.     

Human Semen Refrigeration at + 4 °C: Bio-kinetic Characteristics

The aim of our study was to evaluate the bio-kinetic characteristics of human semen refrigerated for different periods and to compare the effects of refrigeration at +4 °C against cryopreservation of human sperm at −196 °C. Semen was obtained from 30 male partners of infertile couples (infertile subjects) with the following semen profile: sperm count ≥10 × 10⁶/ml; progressive motility ≥20%; atypical forms <70% and white blood cells <1.0 × 10⁶/ml. Fifteen normospermic subjects were also selected as controls (control subjects). The following tests were carried out on basal, refrigerated and cryopreserved sperm: a) sperm kinetic properties (by Superimposed Image Analysis System); b) the Hypoosmotic Viability Test (HVT) (combined Hypoosmotic Swelling and Viability Test). The results of the study showed that the percentage recovery of kinetic properties and of HVT were optimum for up to 48 h. After refrigeration for 72 h, a drastic decrease in straight motility recovery was observed. No significant differences were observed between cryopreservation and refrigeration at +4 °C for 48 h for motility or HVT recoveries in samples from control subjects. However, in infertile subjects, a significant decrease in straight progressive motility and HVT recoveries was observed in cryopreserved samples compared to those refrigerated for 48 h. Neither refrigeration nor cryopreservation led to the growth of pathogenic bacteria in any of the cases studied. Based on the above results, refrigeration could represent a useful alternative to the cryopreservation method.     

Plasma membrane calcium ATPase 4 (PMCA4) co‐ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity

Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca²⁺‐ATPase 4 (Pmca4) gene which encodes the highly conserved Ca²⁺ efflux pump, PMCA4. This is the major Ca²⁺ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co‐immunoprecipitation (Co‐IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca²⁺] in capacitated and Ca²⁺ ionophore‐treated sperm and with neuronal (nNOS) at basal [Ca²⁺] (ucapacitated sperm). FRET efficiencies for PMCA4‐eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4‐nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co‐immunoprecipitated with eNOS in a Ca²⁺‐dependent manner. In Pmca4^?/? sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild‐type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co‐ordinates Ca²⁺ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4^?/? males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance.     

Superovulation and embryo transfer in Holstein cattle using sexed sperm

The use of sexed bull sperm in multiple ovulation and embryo transfer (MOET) programs for Holsteins was evaluated for (1) heifers housed at a commercial embryo transfer (ET) facility (Experiments 1 and 2), and (2) heifers and cows on dairy farms (Experiment 3). In Experiment 1, superstimulated heifers were inseminated with 5 × 10⁶ sexed (X-sorted; n = 5) or unsexed (n = 5) frozen-thawed sperm from one bull at 12 and 24 h after estrus detection. No difference was observed in the rates of transferable embryos (53.4% vs 68.1%), degenerate embryos (24.8% vs 26.6%) and unfertilized ova (21.8% vs 5.3%) between sexed and unsexed sperm, respectively, except for the percent of female transferable embryos diagnosed by embryo sexing (100% vs 49.3%, P < 0.0001). In Experiment 2, donors were inseminated twice with 5 × 10⁶ sexed unfrozen sperm (n = 10) or sexed frozen-thawed sperm (n = 9). Embryo production rates for both treatments were similar to that observed on a commercial ET facility using unsexed sperm. Pregnancy rates for frozen-thawed embryos were similar for sexed and unsexed sperm (70.4% vs 72.4%, respectively). In Experiment 3, 99 flushes were conducted using sexed frozen-thawed sperm from nine bulls but an overall statistical analysis was not completed because the use of bulls was not balanced. However, for one bull with balanced usage, the rate of transferable embryos was higher in heifers than in cows (P < 0.05) inseminated twice with 5 × 10⁶ sperm/dose (10 × 10⁶ total). We concluded that the use of sexed frozen-thawed sperm (≥90% X-sperm biased and 10 × 10⁶ total sperm) may be economically viable for commercial MOET programs in Holstein heifers.     

Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation, colchicine treatment and gene transfection. Results are as follows: (Ⅰ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3% vs. 4.9%, P<0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25μm) as donor nuclei was higher than that from large cells (25-33μm) and small cells (8-15μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS(11.8% vs. 18.6%, P>0.05). (Ⅳ) Fetal fibroblasts treated with 0.05μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P>0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P<0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05μmol/L colchicine could facilitate the development of reconstructed embryos. Additionally, as cells transfected with GFP gene were used as donor nuclei, adverse effect on the development of reconstructed embryos was observed. Therefore, the developmental efficiency of reconstructed embryos could be improved if proper treatments to donor cells were used.     

Cryopreservation with α-tocopherol and Sephadex filtration improved the quality of boar sperm

The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10⁸ sperm; PS: 13.15 ± 2.39 nmol TBARS/10⁸ sperm; S: 13.20 ± 2.18 nmol TBARS/10⁸ sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10⁸ sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10⁸ sperm, PS: 25.61 ± 5.85 nmol TBARS/10⁸ sperm, S: 19.16 ± 3.28 nmol TBARS/10⁸ sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10⁸ sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques.     

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus–oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL⁺) and without (CCL^-) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL⁺), contralateral (CL⁻), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL⁻ cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL⁺ group; however, a higher proportion of embryos were obtained from the CCL⁻ group. Besides, COCs from the CL⁺ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL⁻ group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

In the present study, nuclear transferred embryos (NTEs) were reconstructed by using pig fetal fibroblasts as donors and in vitro matured oocytes as recipients. The effects of G418 selection on donor cells, duration of IVM of prepubertal gilt oocytes and oxygen tension in IVM of oocytes were investigated. The results were as follows: (i) When G418 selected cells expressing GFP were used as donors, the cleavage rate of NTEs decreased drastically in comparison to NTEs derived from donors without antibiotic selection (45.7% vs. 71.6%, p<0.05). For the blastocyst rate, no significant difference was observed between two groups (10% vs. 10.4%, p>0.05). (ii) The rate of nuclear maturation of oocytes increased significantly when IVM duration time was extended from 36h to 42h (83.6% vs. 96.7%, p<0.05). However, no statistical difference were observed between NTEs derived from oocytes of 36 h IVM group and NTEs from oocytes of 42 h IVM group in the rates of cleavage (59.3% vs. 73.6%, p>0.05) and blastocyst formation (9.3% vs. 13.2%, p>0.05); (iii) no significant difference was observed between NTEs reconstructed from oocytes matured under lower oxygen (7% O₂) tension and NTEs derived from oocytes matured under higher oxygen tension (20% O₂) in cleavage rate (70.6% vs. 67.1%, p>0.05) and blastocyst rate (11.8% vs. 12.8%, p>0.05). These results suggest that: (i) G418 selection does not have a significant effect on cleavage rate of NTEs expressing GFP. (ii) Nuclear maturation is greatly improved by prolonging IVM duration from 36 to 42 h, while no significant differences were observed for developmental potential of transgenic embryos. Thus IVM 42 h is the better choice in order to obtain maximum number of MII oocytes as recipients. (iii) Lower oxygen tension and higher oxygen tension in IVM have no significant effect on development of cloned embryos.     

The effect of Tribulus terrestris extract on motility and viability of human sperms after cryopreservation

BackgroundSemen cryopreservation produces significant amounts of reactive oxygen species (ROS), which may lead to impairment of sperm morphology, function, and ultimately, male fertility. Since Tribulus terrestris has antioxidant and free-radical-scavenging properties, this study aims to reveal the effect of the Tribulus terrestris extract on motility and vitality of human sperms after cryopreservation.Materials and methodsSemen specimens from 80 healthy volunteers were divided into eight groups: fresh control (group I), freeze control (group II), groups III, IV, and V, which had 20, 40, and 50 μg/mL doses of Tribulus terrestris extract added before cryopreservation, and groups VI, VII, and VIII, which were supplemented by these extract doses after the freeze-thaw process. To evaluate the effects of the Tribulus terrestris extract, the semen samples were incubated with the extract and evaluated with a light microscope for motility and viability.ResultsAfter cryopreservation, a significant improvement in spermatozoa viability was observed in group VII. In groups VII and VIII, motility, according to World Health Organization (WHO) criteria, increased considerably (p < 0.001). There was no significant difference among groups III, IV, and V.ConclusionThe present study demonstrated that the protective effects of Tribulus terrestris, which improves human sperm motility and viability, may be due to its antioxidant properties. On the basis of the results, the researchers concluded that Tribulus terrestris can be used as a safe therapeutic alternative to current modalities for the management of motility dysfunction in males.     

Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT), combined with genome modification techniques, is a very pow-erful tool for agriculture, medicine and fundamental research on basic biological mechanisms. The effi-ciency of producing transgenic animals is greatly prom…