Embryo outcome in Y-chromosome microdeleted infertile males after ICSI

A prospective study involving 118 infertile Japanese couples to assess the embryo outcomes in both azoospermic and oligoasthenoteratoazoospermic (OAT) patients with Y-chromosome microdeletion. The men were divided into two groups; azoospermia (n = 27), and OAT, sperm concentration <5 x 10(6)/ml (n = 91). They were investigated for Y-chromosome microdeletions by a polymerase chain reaction (PCR) amplification of the Y-chromosome-specific sequence tag site (STS). The embryo outcomes of patients found to have Y-microdeletion were determined. The frequency of microdeletion was 8.8% (9) and two had microdeletions distal to DAZ. The mean fertilization rate and the cleavage rate in the eight cycles of both azoospermic and oligospermic patients were 59.3 and 87.5%, respectively. The percentages of grade 1 & 2 embryos, > or =6 cells embryos, and blastocyts were 51.7, 65.6, and 45.3%, respectively. Three pregnancies resulted from the eight cycles (37.5%). CONCLUSION: in Y-chromosome microdeletion cycles in which sperm cells were available for intracytoplasmic sperm injection (ICSI), embryo outcome was comparable to conventional IVF.     

Sperm function and production of bovine embryos in vitro after swim-up with different calcium and caffeine concentration.

Frozen semen from bulls was used in artificial insemination programs was submitted to swim-up in Sperm Talp media containing different calcium (1.8, 2.6, 3.6 mM) or caffeine (2.5, 5.0, 7.5 microM) concentrations. The following sperm variables were evaluated: sperm recovery, motility, vigor, morphology, alterations in the pattern of capacitation by chlortetracycline (CTC) staining, and alterations in lysophosphatidyl choline (LPC)-induced acrosome reaction (AR). Sperm obtained from swim-up under different conditions were also tested for in vitro embryo production. No significant differences in the variables motility, vigor, morphology, and LPC-induced AR were observed among the treatments. However, the use of caffeine resulted in greater frequency of sperm with the capacitated pattern by CTC staining, compared to controls without caffeine. The greatest frequency of capacitated sperm (53%) was observed with 7.5 microM caffeine. Different calcium and caffeine concentrations in swim-up resulted in no significant differences in the cleavage rate and embryo development. In summary, micromolar concentrations of caffeine in Sperm Talp may stimulate sperm capacitation.     

Sperm-lectin agglutination combined with swim-up leads to an efficient selection of highly motile, viable and heterogeneous ram spermatozoa

Lectins, high molecular weight glycoproteins with different sugar-binding specificity, are able to agglutinate different cell types. The recovery of high-quality spermatozoa can be facilitated by the agglutination induced by the lectin binding. The objective of this study was to combine sperm-lectin agglutination with a dextran/swim-up procedure for developing a new selection technique for ram spermatozoa. To study sperm quality, cell viability (plasma membrane integrity), the HOS-test response and progressive individual motility were assessed. Simultaneously, centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system was carried out to analyze sperm surface heterogeneity. Semen from 3 mature Saltz rams was pooled, and 0.5-mL aliquots were incubated with 4 fluorescein-labelled lectins (ECL, JAC, PSA, RCA). Then, a dextran solution was gently added and overlaid with medium. The top layer of the medium containing the spermatozoa was collected and replaced by careful addition of fresh medium. The incubation sequence was repeated 3 times at 10-min intervals. The consecutive 4 top layers obtained were pooled to give the swim-up combined sample. The highest rate of improvement in sperm quality was obtained after incubation with RCA, with a 50% increase in progressive individual motility, 21.6% in HOS value and 39.5% in viability. Total cell recovery was 64% (1.56x10(9) cells), with a viable cell recovery rate of 86%. The obtained sample showed 82% motility, 80% HOS score and 77% viability, up from the pre-swim-up values of 51, 60 and 57 %, respectively. Comparative CCCD analysis revealed a very high heterogeneous population in the RCA/swim-up sample obtained, while a much more homogeneous population was obtained in the sample after the dextran/swim-up procedure previously developed byus With this simple method, a large proportion of highly-motile spermatozoa with preserved plasma membrane and high heterogeneity can be obtained. These results strongly suggest that this selection procedure could result in a high fertility rate.     

Preparation and recovery of frozen-thawed bovine spermatozoa via various sperm selection techniques employed in assisted reproductive technologies

A number of semen manipulative techniques are currently available to remove the undesirable spermatozoa, debris and other factors and to increase sperm quality. The use of motility stimulants such as caffeine or others could optimize the recovery and quality of frozen-thawed spermatozoa processed by a variety of sperm selection techniques. Frozen-thawed specimens from 5 bulls were slowly diluted and washed with Ham's F-10 medium containing 3% BSA (w/v) and 0 or 2 mM caffeine. Aliquots containing approximately 50 x 10(6) total sperm cells were used for conventional sperm wash, swim-up, Percoll density gradient centrifugation (80, 70, 55 and 40% Percoll gradients) and Sephadex (SpermPrep I) filtration. Quantitative and qualitative characteristics of selected spermatozoa included: total sperm (x 10(6)), percentage and grade (0 to 4) of motility, percentage of spermatozoa with coiled tails and response to the hypoosmotic swelling (HOS) test (percentage of swollen spermatozoa). When compared to washed specimens, fewer spermatozoa were recovered via the swim-up, Percoll and SpermPrep I filtration methods. Quantitative and qualitative characteristics of these spermatozoa improved further after processing with Ham's F-10 containing 2 mM caffeine, followed by selection via the various techniques. Enhancement of sperm motility, in conjunction with the most appropriate sperm selection technique, represents an efficient method for the recovery of spermatozoa with improved qualitative characteristics.     

Integrated morphophysiological assessment of two methods for sperm selection in bovine embryo production in vitro

Extensive work was done regarding the ability of Swim up and Percoll gradient to select functional sperm for in vitro embryo production (IVP) systems. The aim of this work was to compare Swim up and Percoll as methods of sperm selection by ultrastructural, biochemical and functional studies. Frozen-thawed semen from two bulls (Experiments 1 and 2, respectively) were treated using Swim up or Percoll discontinuous gradients. Motility, sperm membrane ultrastructure, sperm proteins, in vitro embryo production (insemination doses, cleavage, embryo yield and quality) and embryo sex ratio were scored and compared. Electron transmission microscopy of outer sperm membranes showed higher (P<0.05) percentage of sperm with lost acrosomes in Percoll treated samples compared to Swim up. A differential protein pattern was also detected. When in vitro embryo production was performed, Percoll gradient produced higher (P<0.05) number of fertilizing doses (7.6 versus 5.9, Bull 1; 13.5 versus 7.8, Bull 2) and higher sperm motility (90% versus 76.6%, Bull 1; 81.7% versus 68.3%, Bull 2) than Swim up. The percentage of cleavage (Day 3) was similar in both treatment groups, whereas embryo production rate (Day 7) was higher (39.4% versus 30.2%, Bull 1; 38% versus 32.4%, Bull 2; P<0.05) when Percoll gradient was used. The percentage of hatched embryos (Day 11) and sex ratio did not differ. Total cell counting and embryo differential staining (inner cell mass and trophoblast cells) of Day 7 embryos showed that Percoll treated sperm produced better quality embryos compared to Swim up. We concluded that Percoll had a better performance selecting sperm and an enhanced capacity for embryo production when compared with the Swim up procedure; this could be attributed to a better acrosome exocytosis, associated to the absence of certain membrane proteins.     

NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by florescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8–10% using naked DNA or liposomes to 40–45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.     

An ICSI rate of 90% minimizes complete failed fertilization and provides satisfactory implantation rates without elevating fetal abnormalities

IVF cycles utilizing the ICSI technique for fertilization have been rising over the 25 years since its introduction, with indications now extending beyond male factor infertility. We have performed ICSI for 87% of cases compared with the ANZARD average of 67%. This retrospective study reports on the outcomes of 1547 autologous ART treatments undertaken over a recent 3-year period. Based on various indications, cases were managed within 3 groupings - IVF Only, ICSI Only or IVF-ICSI Split insemination where oocytes were randomly allocated. Overall 567 pregnancies arose from mostly single embryo transfer procedures up to December 2016, with 402 live births, comprising 415 infants and a low fetal abnormality rate (1.9%) was recorded. When the data was adjusted for confounders such as maternal age, measures of ovarian reserve and sperm quality, it appeared that IVF-generated and ICSI-generated embryos had a similar chance of both pregnancy and live birth. In the IVF-ICSI Split model, significantly more ICSI-generated embryos were utilised (2.5 vs 1.8; p?<?0.003) with productivity rates of 67.8% for pregnancy and 43.4% for livebirths per OPU for this group. We conclude that ART clinics should apply the insemination method which will maximize embryo numbers and the first treatment for unexplained infertility should be undertaken within the IVF-ICSI Split model. Whilst ICSI-generated pregnancies are reported to have a higher rate of fetal abnormalities, our data is consistent with the view that the finding is not due to the ICSI technique per se.     

Potassium movement during sodium-induced motility initiation in the rat caudal epididymal spermatozoa

The sodium-induced sperm motility initiation of the rat cauda epididymal sperm has been studied in different potassium concentrations. High K+ inhibited motility initiation. At a K+ concentration of 50 mM (concentration found in the rat cauda epididymidis), sperm motility was inhibited by 80%. K+ movement across the sperm membrane has been followed by using 86Rb+ as a marker for K+. When the 86Rb+ preloaded sperm were suspended in a sodium-free medium, there was a little efflux of 86Rb+. However, if they were suspended in a sodium-containing medium, the efflux rate was greatly increased. This increase in 86Rb+ efflux rate was associated with an initiation of sperm motility. Both 86Rb+ efflux and motility initiation were triggered by a K+ ionophore 18-crown-6 (2 X 10(-3)M). However, the ionophore-induced 86Rb+ efflux and motility initiation only occurred in the presence of extracellular Na+. Tetraethylammonium (TEA) chloride, which blocks K+ channels, inhibited motility initiation in a dose-dependent manner. Changes in the membrane potential of sperm have been followed using the fluorescent dye diO-C6-(3) whose fluorescence in sperm suspension changes markedly with changes in sperm membrane potential. During motility initiation, there was a fall in fluorescence of the dye due to increased partition into sperm cells. This observation may indicate a hyperpolarization of the sperm membrane during motility initiation. It was concluded that sperm motility initiation is associated with a complex ionic event. Na+ enters sperm cells in exchange with H+ and K+. This change in the permeability of the sperm membrane to ions is reflected by a change in the sperm membrane potential.     

Baboon serum is superior to human or bovine serum albumin for baboon sperm capacitation and zona binding

Background  Baboon in vitro fertilization requires capacitated sperm in appropriate media. In this study, we compared the effect of baboon serum (Bas), human serum albumin (HSA) and bovine serum albumin (BSA) on baboon sperm capacitation.
Methods  Five males (n = 5) were electroejaculated and 43 oocytes retrieved from super-ovulated female baboons (n = 10). Each sperm sample was assessed for initial motility and concentration before and after swim-up. For swim-up, each sperm sample was incubated separately in Biggers–Whitten–Whittingham media containing either BaS, HSA, BSA or without protein supplementation (control). After swim-up, each sperm aliquot was incubated with two to three oocytes. The number of sperm bound to the zona was evaluated after overnight incubation.
Results  Sperm motility and zona binding was significantly higher after capacitation in media supplemented with BaS than in HSA or BSA or in media without protein supplementation ( P  < 0.05).
Conclusion  Baboon serum is superior to HSA or BSA for baboon sperm capacitation and zona binding.     

Embryo recovery rate and recipients’ pregnancy rate after nonsurgical embryo transfer in donkeys

Sixty-three embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%) of five Pantesca jennies, 2 to 5 yr old, naturally mated or artificially inseminated with fresh semen. Embryo recovery rate was influenced by number of ovulations per cycle (133% and 63% for double and single ovulations, respectively), by the day of embryo recovery attempt (12%, 83%, and 75% at Days 7, 8, and 9 after ovulation, respectively), and by the repetition of the embryo recovery attempt on successive cycles (60%, 79%, and 100% for cycles 1 to 7, 8 to 14, and 15 to 24, respectively). All recovered embryos but three were classified as good or excellent. Of 58 nonsurgical embryo transfers to Ragusana jenny recipients, 13 (22.4%), 10 (17.2%), and 9 (15.5%) resulted in a pregnancy at Days 14, 25, and 50, respectively. Recipients’ pregnancy rate was not influenced by the evaluated parameters: embryo quality and age, media employed to wash embryos, days after ovulation of the recipient, experience of the operator. Between 14 and 50 d of pregnancy, 4 of 13 (30.7%) embryos were lost with an influence of the days from ovulation of the recipient: recipients at Days 5 or 6 kept all pregnancies (N = 7), whereas recipients at Days 7 or 8 lost 3 of 4 pregnancies, as one of the two recipients at Day 3. More studies are needed before embryo transfer could be considered a reliable tool to preserve endangered donkey breeds.     

A modified swim-up method reduces polyspermy during in vitro fertilization of porcine oocytes

The general method of porcine in vitro fertilization (IVF), involving the co-culture of both gametes in a medium drop, is thought to be the main reason for the high incidence of polyspermy. The aim of this study was to reduce the polyspermic fertilization of porcine embryos during IVF by the modified swim-up method, based on general sperm swim-up technique. Within this design, a 70 μm pore sized cell strainer was used to separate the sperm pellet placed at the bottom of a tube from the mature oocytes placed within the upper region. The separation of gametes using this permeable barrier was to ensure that only motile sperm gained access to the oocytes. It was found that the rate of polyspermy was significantly lowered for the sperm preparations from three boar breeds in modified swim-up method when compared with that of the general microdrop method (p < 0.05). However, the penetration rates were found to be similar in both methods for two boar breeds. The average occurrence of blastocysts with more total cell number was higher in the modified swim-up method, while no significant difference in blastocyst rates between the two IVF methods was observed. The frequency of normal diploid embryos was also significantly higher in the modified swim-up method and polyploidy was more frequently observed in microdrop method (p < 0.05). Our results demonstrated that the modified swim-up IVF method could reduce polyspermic penetration, and consequently produce better quality and karyotypically normal embryos in porcine IVF.     

Semen characteristics and artificial insemination in rockhopper penguins (Eudyptes chrysocome chrysocome)

This work was performed as part of a multi-year study to determine the cause of the low fertility in captive rockhopper penguins (Eudyptes chrysocome chrysocome) and attempt to increase the fertility through artificial insemination (AI). Semen collection and characterization was performed on 14 male rockhopper penguins. The samples were evaluated for volume, sperm concentration, and sperm quality (motility, forward motility, viability, and morphology). There was a large variation between individuals and between collections for each individual. Mean volume of ejaculate was 0.24 ml. Mean concentration was 47.09 × 10(6) sperm/ml. Mean number of sperm per collection was 6.57 × 10(6). The mean motility was 49.4%. Mean forward motility was 1.7. Mean percentage of living sperm was 88.9%. Mean percentage of sperm with normal morphology was 69.4%. AI was performed on a total of 10 females using pooled semen samples. The birds were also allowed to naturally mate. Ten eggs were laid and three fertile eggs were produced, one of them hatched but died within 24 hr. Paternity testing was performed using 12 microsatellite loci, but unfortunately due to insufficient variability, the paternity of the chick and two embryos could not be determined.     

Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls

The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge.     

Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure^? and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure^? and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure^?, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure^? method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure^? treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure^? method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure^? could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos.     

Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from frozen-thawed buffalo semen

Frozen-thawed semen of five buffalo bulls was used to compare efficacy of swim-up and Percoll gradient methods for separating viable spermatozoa. Sperm separated by the two methods were also tested to differentiate buffalo bulls on the basis of in vitro fertilization (IVF) rates. Recovery of motile sperm (%), increase in membrane integrity (%) and acrosome integrity (%) were compared after two sperm separation methods in experiment I, and in vitro fertilization rate (cleavage rate and cleavage index) was compared in experiment II. Swim-up separated sperm showed a higher motility (P<0.05), while percent recovery of motile sperm was higher with Percoll separation (P<0.05). Membrane integrity (%) of sperm separated with swim-up was significantly higher (P<0.05) as compared to sperm separated with Percoll gradient. Swim-up separated sperm gave a higher cleavage rate and cleavage index (P<0.001). Sperm separated by swim-up showed significant difference among the bulls in cleavage rate and cleavage index (P<0.05), while the Percoll gradient method did not. It has been concluded that separation of sperm from frozen-thawed buffalo semen by swim-up method can be more expedient for IVF in buffalo.     

Evaluation of the vervet (Clorocebus aethiops) as a model for the assisted reproductive technologies

The vervet monkey was evaluated as a primate model for use in assisted reproductive technologies (ARTs). Eight adult female vervets were hormonally monitored for their potential use as egg donors and those six females displaying regular menstrual cycles were subjected to controlled ovarian stimulation with recombinant human gonadotropins. Three animals failed to respond while laparoscopic follicular aspiration was performed on the other three females at 27-30 h post-human chorionic gonadotropin administration. A total of 62, 40, and 18 oocytes was recovered from these three animals of which 30, 20, and 4, respectively, matured to the metaphase II stage and were subsequently inseminated using intracytoplasmic sperm injection. An average of 40+/-15% (SEM) of the inseminated oocytes were fertilized based on pronucleus formation and timely cleavage. One embryo from each of the two stimulated females developed into expanded blastocysts. Two adult male vervets were assessed as sperm donors. Neither adjusted well to the restraint and collection procedure required for penile electroejaculation. Samples collected via rectal electroejaculation were very low in sperm motility and concentration; however, cauda epididymal aspirations from one male yielded an adequate concentration of motile sperm. These results emphasize the need to establish species-specific ovarian stimulation protocols and semen collection techniques if vervets are to be considered for basic and applied (ARTs) research on primate gametes or embryos.     

A dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from seminal plasma

Although washing of sperm cells by centrifugation is a procedure in widespread use, there have been indications that centrifugation may be harmful to the cells. The objective of this study was to develop a modified swim-up technique, without centrifugation, to get a selection of highly motile and viable ram spermatozoa free of semen plasma.Semen collected from 3 rams over a period of a year was pooled into a low, medium and high motility group, and aliquots from each pool were placed beneath a dextran solution and overlaid with medium. The top layer of the medium was collected (and replenished) 4 times at 15-min intervals. Evaluated were the pre- and post-swim-up progressive individual motility, membrane integrity and resistance to a hypoosmotic swelling test (HOS). Semen samples with initial motility < 60% showed the highest relative improvement in all 3 parameters; samples with 65 to 70 and > 70% initial motility improved less but showed final absolute values similar to those in the low motility group and to each other. The first swim-up layer had the highest contamination with semen plasma (17% beta-N-acetylglucosaminidase (NAG), 13% citric acid content) and the lowest motility score. The second, third and fourth fractions were pooled and showed low plasma contamination (2% NAG and 5% citrate), 80% motility, 70% HOS, and 72% viability, up from the pre-swim-up values of 68, 66 and 59%, respectively. Our data suggest that the dextran swim-up procedure is suitable for evaluating ram spermatozoa for in vitro and in vivo procedures in assisted reproduction.     

Effect of donor stimulation, frozen semen and heparin treatment on the efficiency of in vitro embryo production in goats

Investigations on in vitro embryo production in goats in comparison with other domestic species, especially cattle, have been the subject of few reports despite their usefulness for both basic research and commercial application. The objectives of this study were to compare the efficiency of IVP in goats using immature follicular oocytes recovered from FSH-primed and control goats. After IVM, oocytes were fertilized with fresh or frozen-thawed semen capacitated with or without heparin. Mature oocytes were fertilized in vitro with fresh and frozen-thawed sperm of a single buck. Sperm preparation included swim-up separation and heparin treatment (50 micrograms/ml of sperm suspension for 45 min) before spermatozoa were added to oocytes in TALP-IVF. After IVF, the zygotes were cultured for 24h and cleaved embryos were further cultured with goat oviduct epithelial cells or transferred to synchronized recipients. Mean number of oocytes recovered from FSH-primed goats (24.5 +/- 8.6) was significantly higher (P < 0.01; t test) in comparison to control does (14.7 +/- 4.7). Irrespective of fresh or frozen semen, no differences were observed in blastocyst yield when sperm was treated with heparin. However, the highest cleavage rate (99/126; 79.4%) as well as blastocyst yield (47/126; 37.3%) was obtained after IVF with fresh sperm capacitated without heparin. Contrary to fresh sperm, heparin treatment of frozen-thawed sperm significantly improved (P < 0.01) embryo cleavage. No differences between in vivo developmental competence of embryos related to sperm origin were found after transferring into recipients. Overall, more than 60% of the recipients became pregnant and 20% of all transferred embryos survived delivering 13 healthy kids. Our caprine IVP system allows obtaining embryos with developmental competence comparable to bovine IVP.     

Effect of Percoll volume, duration and force of centrifugation, on in vitro production and sex ratio of bovine embryos

The objective was to evaluate the effect of Percoll volume, and duration and force of centrifugation on sperm quality characteristics, embryo development, and sex ratio of in vitro-produced (IVP) bovine embryos. Frozen-thawed semen from four bulls were submitted to three Percoll procedures: T1—4 mL of Percoll, centrifuged for 20 min at 700 g; T2—800 μL of Percoll, centrifuged for 20 min at 700 g; and T3—800 μL of Percoll, centrifuged for 5 min at 5000 g. Sperm total motility, morphology and integrity of the sperm acrosome, membrane and chromatin were determined before and after Percoll treatment, and semen was used for in vitro fertilization (IVF) of in vitro-matured oocytes. All Percoll methods increased the proportion of motile sperm (P < 0.05). There were no significant effects of treatment for any sperm characteristic; however, for every end point, there were significant differences among bulls. Similarly, rates of cleavage and blastocyst formation were not affected by the Percoll procedure (P > 0.05), but were affected by sire (P < 0.05). Sex ratio was similar among treatments for Bulls 2 and 3, whereas semen from Bull 1 processed by T1 yielded a greater percentage of male embryos. However, when only treatments were considered, independent of bulls, the proportion of male:female embryos did not differ significantly from an expected 1:1 ratio. In conclusion, decreasing Percoll volume, reducing duration of centrifugation, and using a higher force of centrifugation did not significantly affect sperm quality, embryo development, or sex ratio of in vitro-produced bovine embryos.     

Embryo quality,and not chromosome nondiploidy,affects mitochondrial DNA content in mouse blastocysts

It has been shown recently that there is premature mitochondria biosynthesis in blastocysts from older women whose egg or embryo quality is poor and that aneuploid blastocysts also have a high number of mitochondrial DNA (mtDNA) copies. Whether nondiploidy/aneuploidy or reduced egg or embryo quality causes premature mitochondrial biosynthesis is not known. This study constructed haploid, diploid, triploid, and tetraploid blastocysts by parthenogenetic activation, intracytoplasmic sperm injection with one or two sperm heads, blastomere electrofusion, respectively, and generated reduced cytoplasm quality embryos from diabetic mouse and in vitro fertilization of aged oocytes, and examined whether nondiploidy or reduced cytoplasm quality causes premature mitochondrial biosynthesis. MtDNA numbers of each blastocyst from different models were tested by absolute quantitative real-time polymerase chain reaction. It was found that mtDNA content in preimplantation embryos was not associated with their chromosome ploidy, while mtDNA copy numbers in embryos with suboptimal quality were increased. Therefore, it might be the reduced cytoplasmic quality, and not chromosome nondiploidy, that causes premature mitochondria biosynthesis in blastocysts.