Elimination of bacteria from human semen during sperm preparation using density gradient centrifugation with a novel tube insert.

The occurrence of bacteria in sperm samples intended for in vitro fertilisation can compromise the outcome of assisted reproductive techniques. Effective semen processing procedures should therefore be implemented to remove bacteria from semen. Unfortunately, technique failure does occur whereby bacteria can be found in processed sperm preparations. To improve the effectiveness of semen processing, a novel centrifuge tube insert was developed to facilitate the layering of density gradients and semen, and to prohibit the re-infection of purified sperm pellets. The purpose of this study was to: (i) determine the prevalence and type of bacteria present in semen of patients participating in the Unit's assisted reproduction program and (ii) evaluate the effectiveness of density gradient centrifugation with the novel tube insert, for the elimination of bacteria and yeast from spiked human semen samples. A survey in 2007-2010 indicated that 50% of semen samples were found to have positive bacterial cultures. Semen processing by means of density gradient centrifugation with the novel tube insert eliminated significantly more in vitro derived (spiked) bacteria and yeast from semen compared to processing without the insert (P < 0.004). Therefore, it is highly recommended that the centrifuge tube insert, ProInsert™, be incorporated into assisted reproductive programs.

Treatment of human sperm with serine protease during density gradient centrifugation.

PURPOSE: Seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective, whereby vertical or horizontal transmission of the infection could occur. Serine proteases have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could negatively impact on sperm parameters. This study investigated the effect of the addition of a recombinant, human-sequence protease (rhProtease) on sperm parameters during density gradient centrifugation. METHODS: (i) Pooled semen samples (n = 9) were split and processed by density gradient centrifugation, with the top density layers supplemented, or non-supplemented with rhProtease at three different concentrations (diluted 2, 10 and 20 times). Sperm parameters were then analysed by flow cytometry and computer-assisted semen analyses. (ii) Semen samples (n = 5) were split and similarly processed using PureSperm® Pro, with rhProtease in the 40 % density gradient layer, or standard PureSperm® not supplemented with rhProtease (Nidacon, International) respectively. The Hemizona assay was then utilized to compare sperm-zona binding post processing. RESULTS: Evaluation of sperm parameters indicated that rhProtease did not, at any of the tested concentrations, have an impact on (i) mitochondrial membrane potential, vitality, motility, or (ii) zona binding potential. CONCLUSION: We report that the addition of rhProtease to density gradients is a non-detrimental approach that could improve the effectiveness of semen processing for the elimination of seminal pathogens, and benefit assisted reproduction outcome.

Comparison of a recombinant trypsin with the porcine pancreatic extract on sperm used for the in vitro production of bovine embryos.

The objective was to compare the effects of treating bovine semen with two trypsin products (the porcine pancreas extract and a recombinant) and a control (no trypsin) on in vitro embryo production. Our hypothesis was that the trypsin treatments would not cause any significant difference in fertilization and embryo development as compared to the control. Semen was washed through a gradient system containing a porcine-origin trypsin, a recombinant bovine-sequence trypsin, or the control (no trypsin). Oocytes (n=3036) were collected from abbatoir-derived ovaries, matured for 24h, and allocated into three groups: porcine trypsin (n=1040), recombinant trypsin (n=972), and control (n=1024). Ova were inseminated with 1 x 10(6) motile sperm/mL and cultured for 18-24h. Thereafter, presumptive zygotes were cultured for 7 days in 50 microL G1/G2 micro-droplets under mineral oil. Overall, sperm motility was lower before than after each treatment (mean of 51.4% versus 70.2%, respectively; P<0.001); however, motility was not significantly different among the three groups (porcine-origin trypsin=68.8%, recombinant trypsin=69.0%, and control=72.6%). Similarly, there was no significant difference among these groups for cleavage rates (70.1, 70.9, and 73.9%), or the number of morula/blastocyst stage embryos (53.4, 53.3, and 48.7%). In conclusion, treatment of bovine sperm with either porcine-origin trypsin or recombinant trypsin prior to insemination had no detrimental effects on in vitro embryo development.

Comparison of two different methods for the vitrification of hatched blastocysts from the dromedary camel (Camelus dromedarius).

The uteri of 32 donor camels were flushed non-surgically on Day 6, 7 or 8 after ovulation and a total of 184 embryos was recovered. Sixty Day 6 embryos and 61 Day 7 embryos were vitrified or frozen ultrarapidly using open pulled straws and a modified version of the Vajta protocol. These embryos were subjected to concentrations of either 10% and 20% or 20% and 40% ethanediol as the cryoprotectant before being loaded into open pulled straws (OPS) and plunged into liquid nitrogen. All embryos were subsequently thawed and rehydrated either directly into holding media or into holding media containing 0.2 M sucrose and were incubated for 5 or 10 min before being transferred to holding media before transfer to recipients. Although the survival rate of the embryos immediately after thawing was high (OPS 20%/40% ethanediol resulted in 97% and 100% survival for Day 6 and Day 7 embryos, respectively; OPS 10%/20% ethanediol resulted in 90% and 70% survival for Day 6 and Day 7 embryos, respectively), after 2 h in culture, survival rates had decreased to 46% and 53% for Day 6 and Day 7 embryos, respectively, using OPS 10%/20% and 53% and 63% for Day 6 and Day 7 embryos, respectively, using OPS 20%/40%; however, none of the embryos transferred resulted in a viable fetus. A further 63 embryos (Day 6: n = 31; Day 7: n = 16; Day 8: n = 16) were subsequently exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 M sucrose + 0.375 M glucose + 3% polyethylene glycol) in three steps and after loading into 0.25 mL straws were plunged into liquid nitrogen. However, a much greater percentage of the Day 7 and Day 8 embryos (43.8% and 81.2% respectively) were fractured or torn after warming and none of the 12 intact embryos transferred resulted in a pregnancy. Better survival rates immediately after thawing and rehydration were obtained with the smaller Day 6 embryos (94%), which resulted in a total of eight fetuses from the 21 embryos transferred.

Developmentally related changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in vitro.

The metabolism of, and retention of radioactivity from, radiolabelled glucose, glutamine and pyruvate were measured in individual cattle embryos produced in vitro from the 2-cell to hatched blastocyst stage. Uptake was defined as the numeric sum of metabolism and retention of radiolabel. Glucose metabolism increased significantly between the 8- and 16-cell stages, but was accompanied by a much larger increase in glucose uptake. Consequently, the proportion of glucose uptake that was metabolized through the pentose-phosphate and Embden-Meyerhof pathways reached a minimum at those stages. From the compacted morula stage onward, the calculated uptake of [14C]glucose was only 25 to 33% of that calculated for [5-3H]glucose. This suggests that 66 to 75% of glucose carbon leaves the embryo, after metabolism to phosphoenolpyruvate, in some form other than CO2. Little or no glucose metabolism by the Krebs cycle could be detected at any stage. Both glutamine and pyruvate metabolism were relatively high at the 2- and 4-cell stages, declined to a minimum at the compacted morula stage and then increased with blastulation. Glutamine metabolism continued to increase with expansion and hatching of the blastocyst, but pyruvate metabolism did not. This suggest that, relative to the activity of the pathway from pyruvate to 2-oxoglutarate, the activity of the 2-oxoglutarate-to-oxaloacetate segment of the Krebs cycle is of increasing significance during expansion and hatching of the cattle blastocyst.

Developmental competence in vitro and in vivo of cryopreserved, hatched blastocysts from the dromedary camel (Camelus dromedarius).

The present paper describes experiments designed to investigate methods for cryopreserving embryos from dromedary camels. Because preliminary studies had shown ethanediol to be the best cryoprotectant to use for camel embryos, the current experiments were performed to determine the minimum exposure time to 1.5 m ethanediol required to achieve cryoprotection. The uteri of 30 donor camels were flushed non-surgically 8 days after mating. Embryos were recovered and 158 were assigned to one of three groups, which were exposed to 1.5 m ethanediol for either 10 min (n = 67), 5 min (n = 51) or 1 min (n = 40). Embryos were subsequently thawed and rehydrated by expelling either directly into holding medium (HM; HEPES-buffered Tyrode's medium containing sodium lactate and 3 mg mL(-1) bovine serum albumin, 10% fetal calf serum, 100 IU mL(-1) penicillin G, 100 microg mL(-1) streptomycin and 25 microg mL(-1) amphotercin B) or initially into HM containing 0.2 m sucrose for 5 or 10 min. The survival rate of all embryos immediately post-thawing, as judged by the morphological appearance of the embryos, was high (91%), but was greatly reduced after 2 h culture (59%). Ninety-two embryos were transferred to recipient camels resulting in 18 viable fetuses (1 min ethanediol exposure, n = 1/15; 5 min ethanediol exposure, n = 3/34; 10 min ethanediol exposure, n = 14/43). Of the embryos rehydrated directly in HM, six of 65 resulted in viable fetuses and those rehydrated initially in 0.2 m sucrose for 5 or 10 min resulted in nine of 47 and three of 46 fetuses respectively. From these experiments, we conclude that camel embryos can be cryopreserved using ethanediol as a cryoprotectant when the embryos are cooled slowly (to 33 degrees C) before being plunged into liquid nitrogen for storage.

Advances in reproduction in captive, female great apes: Value of biotechniques.

Most of the progress in female great ape reproduction has focused on monitoring ovarian and endocrine activity. Perhaps of more importance has been the gradually evolving interest and willingness to consider biotechnology as a potential, viable approach for enhancing reproductive performance. Artificial insemination (AI), in vitro fertilization (IVF), and embryo transfer offer an array of possibilities for combating infertility and understanding the fundamental differences and similarities among great ape species. Multidisciplinary efforts have assessed reproductive competence, from the simple (i.e., perineal tumescence and urinary occult blood in chimpanzees) to the complex (i.e., ovum recovery and IVF following exogenous hormone treatment in chimpanzees and gorillas). Hysterosalpingography and laparoscopy have been used to determine uterotubal patency and to identify pathological conditions in gorillas. Assays for steroid metabolites in serial urine samples, to permit accurate assessments of ovarian cyclicity, have been developed and validated for all great apes species. Stimulation of follicular recruitment and maturation has been achieved following administration of clomiphene citrate (chimpanzees and gorillas) and human menopausal gonadotropin (gorillas). Clomiphene citrate and human chorionic gonadotropin have been used to regulate ovulation for the AI of chimpanzees. Pregnancies have resulted in chimpanzees and gorillas following AI using fresh and cryopreserved/thawed semen; however, conception rates vary. Embryos have been nonsurgically recovered from chimpanzees after timed matings and follicular oocytes have been recovered from chimpanzees and gorillas by transabdominal laparoscopy after exogenous hormone treatment. To date, although in vivo matured ova have been fertilized in vitro using homologous sperm in chimpanzees and gorillas, no great ape offspring have been born from transferred embryos produced by in vitro or in vivo fertilization. A review of the many remaining problems suggests the need for more basic studies of ovulation induction, ovum and sperm requirements in vivo and in vitro, and the effects of animal manipulation (anesthesia, surgery, and other stressors) on the ability of the female to ovulate and sustain pregnancy. Especially important are more assessments to identify reproductively competent individuals so that these females can be placed with breeding males while subfertile or infertile animals are designated for intensive artificial breeding research.

Pronucleus formation following in vitro fertilization of oocytes recovered from a gorilla (Gorilla gorilla gorilla) with unilateral endometrioid adenocarcinoma of the ovary.

A 33-year-old, nonreproductive female gorilla was scheduled for ovariohysterectomy after diagnosing endometrioid adenocarcinoma of the right ovary; the contralateral ovary appeared small and inactive. Follicular recruitment and maturation were stimulated on the left ovary using human menopausal gonadotropin and human chorionic gonadotropin therapy. Three oocytes were recovered and inseminated using a thawed epididymal semen sample collected postmortem and cryopreserved. At 18 h postinsemination, one ovum was fertilized, the second showed evidence of polyspermia, and the third was unfertilized; no further embryonic development was observed. These results demonstrate that viable oocytes can be salvaged from a nonreproductive gorilla using a human exogenous gonadotropin treatment protocol and fertilized in vitro using cryopreserved/thawed epididymal gorilla semen.

Economical and ecological importance of indigenous livestock and the application of assisted reroduction to their preservation.

Among the many mammalian species that are threatened as the result of habitat destruction are numerous species of rare or little-known native livestock that possess features that render them ideally adapted to their environment. Because of the vital and valuable role many of these species play both to the ecology and economy of their native countries, attention is being directed towards initiating breeding programs that might insure their continued survival. This review introduces and highlights the importance of some of these indigenous species and outlines efforts currently underway to apply assisted reproductive technologies to their conservation.

Compromised development of calves (Bos gaurus) derived from in vitro-generated embryos and transferred interspecifically into domestic cattle (Bos taurus).

Advanced reproductive technologies, incuding IVF and interspecies embryo transfer, are becoming increasingly important for the preservation of endangered species. Previous attempts at interspecies transfers between Bos gaurus and Bos taurus have yielded compromised offspring. The goal of this investigation was to characterize the effects of interspecies transfer of IVF-derived embryos on subsequent neonatal outcome. To achieve this goal, fresh Bos gaurus IVF-derived embryos were transferred into Holstein (Bos taurus) recipients. Four fetuses were carried to term. Calf weight, temperature, heart rate, and respiration rate were recorded after birth. Blood samples also were obtained for determination of blood glucose, pH, packed cell volume (PCV), total hemoglobin (tHB), PO2, and PCO2. After parturition, milk production and health status of the recipients were recorded. Two calves were alive at birth, and two calves were stillborn. One of the calves that was born alive died within minutes after birth, while the other lived until approximately 26 h of age. Blood samples obtained from the calf that lived for 26 h showed it to be extremely acidotic and hypoglycemic; this calf also had marked difficulty thermoregulating. At necropsy, all calves showed evidence of in utero gasping and hypoxia, suggestive of premature placental separation. None of the recipient cows showed typical signs of impending parturition. After parturition, lactogenesis in all recipient cows was markedly decreased. On gross examination, placentae resulting from the interspecies transfers had fewer cotyledons that were also much larger in size compared to cotyledons from normal gaur placentae. Calves in this study had abnormalities consistent with those noted from previous interspecies transfers and with IVF and nuclear transfer (cloned) calves. Due to the design of this study, it is not possible to differentiate between problems resulting from the IVF process and those resulting from potential interspecies incompatibilities. However, interspecies transfers of in vitro-produced gaur embryos into Bos taurus are strongly discouraged.

Validation of a reverse transcription nested polymerase chain reaction (RT-nPCR) to detect bovine viral diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells.

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-131) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.

Detection of Campylobacter or Salmonella in turkey semen and the ability of poultry semen extenders to reduce their concentrations.

Campylobacter and Salmonella are the most commonly reported pathogens causing foodborne illness in the United States. In turkeys, the potential that semen used for artificial insemination is contaminated with these foodborne pathogens has not been investigated. Because semen on turkey farms is pooled and then used to inseminate multiple hens, contaminated semen could easily spread these bacteria throughout entire flocks via artificial insemination. The objectives of this study were to 1) determine if semen from commercial turkey farms contained these foodborne pathogens and 2) if present, evaluate the efficacy of semen extenders to reduce or eliminate Campylobacter and Salmonella from semen. Semen was collected from randomized pools of ejaculates from 10 to 30 toms per farm from 6 flocks over a 7-wk period and, on occasion, was found to contain Campylobacter, Salmonella, or both. To evaluate the efficacy of semen extenders to reduce or eliminate pathogens, pooled ejaculates were challenged with Campylobacter or Salmonella and treated with commercial poultry extenders containing various concentrations of antibiotics or an antibiotic combination previously demonstrated to remove Campylobacter from mammalian semen. Results demonstrate that commercial turkey semen may contain Campylobacter or Salmonella, and the semen extenders tested either did not reduce the bacteria or reduced but did not eliminate these bacteria from semen. We concluded that semen may be a potential vehicle for Campylobacter transfer to hens, and, if this is true, development of a method for eliminating pathogens in semen before insemination could reduce the risk of colonization.

Production of four identical calves by the separation of blastomeres from an in vitro derived four-cell embryo.

The blastomeres of two in vitro derived four-cell embryos were separated and transferred individually into surrogate zonae pellucidae, then co-cultured with bovine oviductal epithelial cells for five days until blastulation. Pairs of the quarter blastocysts were co-transferred with trophoblastic vesicles into each of four synchronised Holstein heifers, three of which were diagnosed pregnant at 28 days gestation, carrying twin fetuses. Four genetically identical bull calves were delivered by elective caesarean section at term pregnancy. One pregnancy was terminated at 56 days.

In vitro maturation and fertilization of oocytes recovered from free-ranging Burchell's zebra (Equus burchelli) and Hartmann's zebra (Equus zebra hartmannae).

A noninvasive repeatable method to harvest oocytes for in vitro fertilization (IVF) could potentially be used to assist reproduction in endangered equid species. The objectives of this study were to evaluate a specific transvaginal ultrasound-guided oocyte recovery procedure for use in zebra mares and the general applicability of IVF procedures in zebra. Ovaries were collected from Burchell's zebra (Equus burchelli) and Hartmann's zebra (Equus zebra hartmannae) mares at routine culling for Expt. I. Of the 144 oocytes recovered from these ovaries, 70% were of excellent quality. No significant difference in oocyte quality was found between the two zebra species. Zona drilling was performed on in vitro-matured oocytes prior to IVF. Epididymal sperm from culled Burchell's zebra stallions were used for IVF. The sperm either were exposed to calcium ionophore or were not treated and served as a control. In vitro fertilized oocytes were then co-cultured with zebra granulosa cells (ZGC) or with bovine oviduct cells (BOC) for up to 8 days. Overall, a 38% cleavage rate was obtained with 16% of sperm-exposed oocytes developing to the morula or blastocyst stage. All of the embryos that developed to at least the morula stage were cultured on BOC; whereas, none of those cultured on ZGC reached the morula stage during the same interval. Cleavage rates of oocytes inseminated with ionophore-treated or with control sperm were not significantly different, suggesting that ionophore treatment of epididymal sperm for IVF in these zebra species may be redundant. In Expt. II, 10 transvaginal ultrasound-guided oocyte aspiration procedures on five captive Burchell's zebra mares recovered a total of 33 oocytes (6.6 oocytes/female) of which 94% were considered viable. This approach may be an attractive means of producing gametes for assisted reproduction in endangered species. Furthermore, results from this study indicate that IVF may become a means of producing offspring from zebra and other equid species in the future.

Natural and gonadotropin-induced ovarian activity in tigers (Panthera tigris) assessed by fecal steroid analyses.

Fecal samples were collected from female tigers (n = 17) to compare endocrine patterns associated with natural ovarian activity with those after chorionic gonadotropin ovulation induction and artificial insemination (AI). Baseline fecal estradiol concentrations were 65.77 +/- 3.61 ng/g with estrual peaks of 167.39 +/- 9.92 ng/g and an anovulatory cycle length of 17.96 +/- 0.70 days. Peak fecal estradiol was higher when females were housed with a male for breeding (262.30 +/- 41.43 vs. 165.30 +/- 3.67 ng/g; P < 0.05). The majority of animals showed some seasonal differences in fecal estradiol however, patterns were inconsistent. Fecal progestagens increased only after breeding confirming tigers are primarily induced ovulators. The non-pregnant luteal phase was 34.50 +/- 1.85 days in duration. In pregnant tigers, fecal progestagens remained elevated for 108 days until parturition and the diagnosis of pregnancy was possible based on the elevated fecal progestagens after 35 days of gestation. Tigers were administered equine chorionic gonadotropin (eCG) to stimulate follicular growth and human chorionic gonadotropin (hCG) to induce ovulation prior to AI [200 IU eCG/100 IU hCG (n = 5); 400 IU eCG/200 IU hCG (n = 2); 500 IU eCG/100 IU hCG (n = 2); 1000 IU eCG/750 IU hCG (n = 11); 1000 IU eCG/1000 IU hCG (n = 4)]. None of the tigers subjected to AI became pregnant (n = 9). Fecal endocrine patterns in gonadotropin-stimulated tigers were considerably different from those observed in naturally bred tigers. In particular, fecal estradiol concentrations were higher than those observed during natural estrus and remained elevated for longer periods of time in tigers administered the higher doses of gonadotropins typically used in conjunction with AI in this species. These abnormal endocrine patterns may help explain the poor success rate of AI in this species.

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla).

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa.

Changes in the metabolism of glucose, pyruvate, glutamine and glycine during maturation of cattle oocytes in vitro.

After maturation in vitro for 0, 6, 12, 18 or 24 h, the metabolism of radiolabelled glucose, glutamine, pyruvate and glycine by individual cattle oocytes was measured for 3 h. The metabolism of glucose through the Embden-Meyerhof (1.77-2.66 pmol per oocyte per 3 h) and pentose-phosphate (0.39-0.75 pmol per oocyte per 3 h) pathways was low and did not change over time. The oxidative metabolism of glucose carbon through the Krebs cycle was low throughout maturation, but increased significantly (P < or = 0.05) at 6 h (0.41 pmol per oocyte per 3 h) and 18 h (0.69 pmol per oocyte per 3 h). Pyruvate, glutamine and glycine metabolism in the Krebs cycle increased during culture. Pyruvate metabolism increased significantly from 0 h (17.3 pmol per oocyte per 3 h) to 6 h (23.3 pmol per oocyte per 3 h) and reached a maximum at 12 h (30.8 pmol per oocyte per 3 h). Glutamine metabolism was unchanged from 0 to 12 h (0.89 pmol per oocyte per 3 h), and then increased significantly at 18 h (2.25 pmol per oocyte per 3 h). Glycine metabolism increased significantly from 6 h (0.21 pmol per oocyte per 3 h) to 12 h (0.46 pmol per oocyte per 3 h) and reached a maximum at 18 h (0.68 pmol per oocyte per 3 h). The results suggest that oxidative metabolism increases, and is the major site of cellular energy production, during maturation of the cattle oocyte in vitro.

Intraspecific blastocyst reconstitution in mice using giant trophectodermal vesicles produced by multiple embryo aggregation.

Giant trophectodermal (TE) vesicles, produced by aggregating multiple embryos, were evaluated as a means of enhancing the viability of reconstituted murine blastocysts. Previous studies have indicated that the development of chimeric conceptuses produced by multiple embryo aggregation is impaired, thus the initial objective here was to examine how this is affected by 1) differences in developmental rates of individual embryos comprising the aggregates, 2) lengths of in vitro culture, and 3) incompatibilities between maternal hosts and chimeric fetal allografts. The results indicate that the viability of aggregates was influenced by embryo genotype and length of in vitro culture. The average number of B6D2-F1 x BL/6 offspring resulting from aggregates cultured for 0.7 day was not significantly different (p greater than 0.05) from that obtained from either embryo singletons cultured for 0.7 and 1.7 days (2.9 +/- 0.3 and 3.9 +/- 0.4, respectively) or from nonmanipulated controls (3.2 +/- 0.2 and 4.3 +/- 0.3, respectively). Blastocyst reconstitution studies were then conducted using giant TE vesicles from B6D2-F1 x BL/6 mice and inner cell masses from C3H mice. The average number of C3H offspring produced (2.7 +/- 0.1) was not significantly different (p greater than 0.05) from that of B6D2-F1 x BL/6 embryo aggregates (3.2 +/- 0.6) or of nonmanipulated controls (4.3 +/- 0.3). The results demonstrate that giant TE vesicles can be effectively used for intraspecific blastocyst reconstitution in mice.

Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and co-cultured in vitro.

The metabolism of radiolabelled glucose and glutamine was measured in individual cattle embryos produced by in vitro maturation and fertilization of oocytes, and culture with bovine oviductal epithelial cells. Metabolism of glucose through the pentose-phosphate pathway increased almost 15 times and the total metabolism of glucose 30 times, during development from the two-cell to the expanded blastocyst stage. The first marked increase in glucose metabolism did not occur until between the eight- and 16-cell stages, the time of activation of the embryonic genome. Conversely, the metabolism of glutamine was high in two- and four-cell embryos and then decreased to reach a minimum at the compacted morula to blastocyst stage, possibly because of degradation of maternally derived enzymes. Blastocyst expansion was accompanied by significant increases in the metabolism of glucose and glutamine, presumably reflecting the increased energy demands of Na(+)-K+ ATPase necessary for formation and maintenance of the blastocoel.

Ultrastructure of oocyte maturation, fertilization, and early embryo development in vitro in the Siberian tiger (Panthera tigris altaica).

The application of assisted reproduction techniques to wild cats has been stalled by a lack of basic knowledge of the reproductive biology in these species. In this study, the ultrastructure of Siberian tiger (Panthera tigris altaica) cumulus-oocyte-complexes (COCs), as well as in vitro produced (IVP) zygotes and embryos were investigated, to estimate the normality of the manipulated reproduction processes. Adult female tigers were subjected to a purified porcine pFSH/pLH stimulation treatment followed by oocyte aspiration. According to morphological appearance at the stereomicroscopical level, COCs were classified as mature, immature, or degenerated, and then allocated into the following groups: presumptively immature COCs, which were in vitro matured (IVM-group) before fixation; presumptively mature COCs, which were either fixed after retrieval (pre-IVF-group), following in vitro insemination (IVF-group) or following in vitro insemination and subsequent in vitro culture (IVC-group). All specimens were processed for light and transmission electron microscopy (TEM). Both the IVM- and pre-IVF-group included oocytes in meiotic stages ranging from prophase I to metaphase II, and some prophase I oocytes in the IVM-group were apparently in their growth phase. The IVF-group presented features of presumptive normal fertilization, but aberrations such as polynucleation were also noted. The IVC-group included cleavage stage embryos of which, however, many were polynucleated. In conclusion, the procedures used for stimulation, aspiration, and classification of COCs resulted in retrieval of a heterogeneous population of oocytes which, following IVF and IVC, displayed a high rate of developmental deviations.