Incidence of abnormal offspring from cloning and other assisted reproductive technologies.

In animals produced by assisted reproductive technologies, two abnormal phenotypes have been characterized. Large offspring syndrome (LOS) occurs in offspring derived from in vitro cultured embryos, and the abnormal clone phenotype includes placental and fetal changes. LOS is readily apparent in ruminants, where a large calf or lamb derived from in vitro embryo production or cloning may weigh up to twice the expected body weight. The incidence of LOS varies widely between species. When similar embryo culture conditions are applied to nonruminant species, LOS either is not as dramatic or may even be unapparent. Coculture with serum and somatic cells was identified in the 1990s as a risk factor for abnormal development of ruminant pregnancies. Animals cloned from somatic cells may display a combination of fetal and placental abnormalities that are manifested at different stages of pregnancy and postnatally. In highly interventional technologies, such as nuclear transfer (cloning), the incidence of abnormal offspring continues to be a limiting factor to broader application of the technique. This review details the breadth of phenotypes found in nonviable pregnancies, together with the phenotypes of animals that survive the transition to extrauterine life. The focus is on animals produced using in vitro embryo culture and nuclear transfer in comparison to naturally occurring phenotypes.

Depletion of testis cell populations in pre-pubertal Bos indicus cattle by irradiation.

Recovery of spermatogenesis following a single dose of irradiation was evaluated in pre-pubertal Brahman bulls, after receiving a single dose of 3, 6, 9 or 12Gray (Gy) irradiation. Biopsy samples of testis tissue were collected and processed for immunohistology at various times following irradiation. Spermatogenic recovery was defined by the changes in tubule diameter, and absolute numbers of undifferentiated spermatogonia (PLZF positive cells) and Sertoli cells (GATA-4 positive cells) per tubule cross section. The effect of irradiation on the depletion of testicular cells was dose-dependent. Immunohistological results from both the 9 and 12Gy group showed degeneration of seminiferous tubules, compared with other doses and controls. From 2 weeks after the treatment, irradiation resulted in a significant and dramatic reduction in tubule diameter (up to 40%), number of undifferentiated spermatogonia (up to 90%) and Sertoli cells (up to 70%), which was sustained for up to 16 weeks post-irradiation in 9 and 12Gy groups (P<0.0001). However, a moderate depletion effect was observed in the 6Gy treatment groups, compared with 9 and 12Gy doses. The 6Gy treatment had significant effects on spermatogonia (up to 79% reduction) and Sertoli cell (30% reduction) numbers following irradiation (P<0.0001). In contrast, the 3Gy dose had no significant effect at either 3 or 5 weeks post-irradiation on tubule diameter, spermatogonia or Sertoli cells. In conclusion, the results from the current study suggest that treatment of recipient testes with a single dose of 6Gy irradiation can temporarily deplete spermatogonial cells in pre-pubertal Brahman bulls, whilst minimising the impact on Sertoli cells and tubule morphology.

Size distribution of methylmercury associated with particulate and dissolved organic matter in freshwaters.

Water samples were collected from 20 wetland, river and lake sites across Eastern Ontario and Western Quebec to investigate the distribution of methylmercury (MeHg) associated with various size fractions of dissolved organic matter (DOM). Tangential Flow UltraFiltration (TUF) was used to fractionate DOM by nominal molecular size (<0.2 microm, <300 kDa, <30 kDa, <5 kDa and <1 kDa). DOM fluorescence (DOM FL) and absorbance (DOC Abs) were used to quantify DOM photoreactivity and aromaticity in each sample. Significant differences in the size-associated distribution of MeHg, Dissolved Organic Carbon (DOC), DOM FL, and DOM Abs were observed between wetlands, rivers, and lakes. The low molecular weight (LMW) fraction (<5 kDa) in wetlands contained the majority of MeHg (70.0+/-13.8%), DOC (56.1+/-9.4%), and DOM FL (77.4+/-7.5%). DOM FL was also high in the LMW fraction for rivers (60.6+/-25%) and lakes (75.2+/-16.9%). Mean MeHg concentrations in the LMW fraction of lakes (41+/-26 pg L(-1)) and rivers (32+/-19 pg L(-1)) were substantial but much lower than wetlands. Rivers had the highest percentage of methylmercury (38.0+/-23.5%) in the particulate (>0.2 microm) fraction. This research highlights the importance of low molecular weight dissolved organic matter in methylmercury fate. For example, a large proportion of MeHg was found in the LMW weight fractions (mean=47.3+/-25.4%) of the wetlands, rivers, and lakes in this study.

Irradiation enhances the efficiency of testicular germ cell transplantation in sheep.

Testis germ cell transplantation in livestock has the potential for production of transgenic genotypes and for use as an alternative to artificial insemination in animal breeding systems. In a pilot experiment, we investigated a workable protocol for testis germ cell transplantation in sheep, including donor cell isolation, rete testis injection, and microsatellite detection of donor spermatozoa in recipient semen. In a second experiment, the effect of depletion of endogenous stem cells with a single irradiation dose of 9 Gy (n = 5) or 15 Gy (n = 5) on the outcome of germ cell transplantation was investigated. Irradiation of recipient testes with a single dose of 15 Gy, followed by transplantation 6 wk after depletion, may be most advantageous because it resulted in all recipients (five of five) producing donor-derived spermatozoa, while the 9-Gy and control groups had limited success rates (two of five and one of three, respectively). Using microsatellite markers to detect the presence of donor DNA, 10 rams were identified that produced spermatozoa of donor origin. The proportion of donor DNA was between 1% and 30% of total ejaculate DNA. When three of these positive rams were used in breeding experiments, four donor-derived offspring (four of 50 [8% of progeny])resulted from a recipient in Merino to Merino transplantation. Six lambs (six of 41 [15% of progeny]) were sired by donor-derived Border Leicester sperm produced in a Merino recipient ram; however, no donor-derived offspring were detected among 34 progeny from a second Border Leicester to Merino combination. These results confirm that preparation of recipient animals with a correct dose of irradiation not only enhances the success rate of the transplantation procedure but also increases the proportion of donor spermatozoa in recipient semen. This study represents the first report of the production of live progeny following testis germ cell transplantation using irradiated recipients in a livestock species.

A comparison of methods for preparing enriched populations of bovine spermatogonia.

The objective of the present study was to identify an efficient and practical enrichment method for bovine type A spermatogonia. Four different enrichment methods were compared: differential plating on laminin- or Datura stramonium agglutinin (DSA)-coated flasks, percoll-gradient isolation, magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The isolated cells were characterised with Dolichos biflorus agglutinin (DBA) lectin staining for type A spermatogonia and vimentin-antibody staining for Sertoli cells. A 2 x 2 factorial design was used to investigate the enrichment efficiency on laminin and DSA. In the laminin-enrichment groups, 2 h incubation in plates coated with 20 microg mL(-1) laminin yielded a 3.3-fold increase in DBA-positive cells in the adherent fraction, while overnight incubation in flasks coated with 20 microg mL(-1) DSA produced a 3.6-fold increase in the non-adherent fraction. However, the greatest enrichment (5.3-fold) of DBA-positive cells was obtained after 2 h incubation in control flasks (coated with bovine serum albumin). Percoll-gradient centrifugation yielded a 3-fold increase in DBA-positive cells. MACS results showed a 3.5- to 5-fold enrichment while FACS produced a 4-fold increase in DBA-positive cells. It is concluded that differential plating is a better method of recovering large numbers of type A spermatogonia for germ cell transplantation, while MACS or FACS can provide highly enriched viable type A spermatogonia for in vitro culture. Further, the combination of differential plating and other enrichment techniques may increase the purification efficiency of type A spermatogonia.

Microsatellite detection of donor-derived sperm DNA following germ cell transplantation in cattle.

Although autologous and heterologous transplantation has resulted in colonisation of recipient testes in cattle, the ability of the transplanted spermatogonial stem cells to complete spermatogenesis has not yet been determined. The objective of the present study was to identify and validate microsatellite markers that can distinguish the genotype of different individuals and therefore can be used to detect the presence of donor DNA in recipient semen samples. In a previous study by this group, successful colonisation of recipient testes by heterologous transfer using a fluorescent dye was shown. In the present work, some of the same recipient animals were investigated further to monitor donor-derived sperm production. The bovine microsatellite detection method was developed specifically to test the ejaculates of the recipients and can also be used to pre-match individuals before germ cell transplantation. Semen was collected from the recipients 52-98 weeks after transfer and the presence of donor DNA in the samples was determined using microsatellite markers. In one of the recipients, all collected semen samples were shown to be positive for donor-derived cells; however, the percentage of donor spermatozoa in the recipient ejaculate declined with time. The donor DNA was also detected in both single cell suspensions and testis tissue from this recipient. These results demonstrate for the first time that testicular germ cell transplantation between different breeds of cattle is feasible and the recipients thereof are able to produce spermatozoa of donor origin. This technology has potential applications in livestock breeding systems and may provide an alternative to artificial insemination.

Differential proteomic analysis of bovine conceptus fluid proteins in pregnancies generated by assisted reproductive technologies.

Proteomic analysis of bovine conceptus fluid proteins during early pregnancy has the potential to expose protein species indicative of both the overall health of the fetal-maternal environment and fetal developmental status. In this study, we examined the differential abundance of bovine conceptus fluid proteins (5-50 kDa fraction) from naturally conceived, in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT)-derived pregnancies at days 45 and 90 of gestation. In day 45 allantoic fluid (AllF) samples, an atypical cluster of low molecular weight ( approximately 14-16 kDa), low pI (between 3.0 and 4.5 pH units) protein species was increased in three of four IVF samples (30-100-fold increase in protein spot volumes compared to normal). These proteins were identified as paralogs of the bovine cathelicidin antimicrobial protein (CAMP) by MALDI-TOF MS peptide mass fingerprint and MALDI-TOF MS/MS peptide sequence analysis. Peptidoglycan recognition protein and serine (or cysteine) proteinase inhibitor clade B1, were also significantly increased in the corresponding IVF samples. In two of four SCNT AllF samples, a 2-10-fold increase in CAMP protein spot volumes were detected. No aberrant abundance levels of individual protein species were observed in amniotic fluid samples, or in day 90 IVF AllF samples. Identification of unique protein species present in the normal bovine AllF proteome at day 45 is also reported.

Short-term maternal psychological stress in the post-conception period in ewes affects fetal growth and gestation length.

Fetal development can be influenced by maternal environment in the peri-conceptional period. This study investigated the effect of maternal feed intake and psychological stress within the first 6 days after conception on embryo development and fetal growth. Superovulated ewes (n=40) were artificially inseminated with semen from one ram. Ewes were then divided into four groups (n=10): group 1 (control) was fed at maintenance level, group 2 (high) at 2x maintenance, and group 3 (low) at 0.5x maintenance on days 2-6 after conception. Group 4 (stress) was fed at maintenance level and then an intense physical and psychological stress challenge was applied for 1 h only on days 2 and 3 after conception. Embryos were recovered at day 6. A total of 113 transferable grade embryos were transferred singly into synchronized untreated recipients, while the remaining embryos (n=165) were fixed and stained for cell counts. Post-conception maternal stress or feed intake did not alter the cell count or grade of day 6 embryos. Fetuses from the stress group had longer crown-rump lengths at day 30 and longer femur length at day 58. Fetuses from the stressed and high feed groups had greater abdominal circumferences at day 85. Subsequent birth weights were not significantly different. Ewes carrying lambs from the stress treatment had shorter gestation lengths. These results show that short-term perturbations of the post-conception maternal environment have measurable effects on fetal development and gestation length.

Proteomic analysis of bovine conceptus fluids during early pregnancy.

A proteomic analysis of bovine amniotic and allantoic fluids collected around Day 45 of gestation was performed using gel-based and LC-based MS workflows. A depletion/enrichment protocol using ultrafiltration under denaturing and reducing conditions produced an enriched fraction containing protein species predominantly between 5 and 50 kDa molecular weight. The analyses of conceptus fluid proteins were performed using two strategies; first, 2-DE coupled with MALDI-TOF-MS/MS and LC-ESI-MS/MS analysis of individual protein spots and second, a global protein snapshot of the enriched 5-50 kDa protein fraction by LC-ESI-MS/MS and LC-MALDI-TOF-MS/MS. Allocation of bovine specific protein identities was achieved by searching the Interactive Bovine In Silico SNP (IBISS) and NCBInr protein sequence databases resulting in the confident PMF identification and MS/MS confirmation of >200 2-DE generated allantoic fluids protein spots (74 individual protein species identified) and the MS/MS peptide identification of 105 LC-ESI-MS/MS generated protein identities. In total, the identity of 139 individual protein species from allantoic fluids was confirmed with peptide sequence probability MOWSE scores at the p<0.05 level or better. The comparison of bovine Day 45 amniotic and allantoic fluids protein profiles revealed differences between these two conceptus fluids in early pregnancy.

Conceptus-related measurements during the first trimester of bovine pregnancy.

In order to determine the variability inherent in conceptus-related measurements in first trimester bovine pregnancies, conceptus and fetometric parameters from beef cattle pregnancies (n=103) estimated to be between Days 36 and 103 of gestation were examined. During this period, the protein concentration of amniotic fluid ranged between 0.181 and 0.501mg/mL. The amniotic fluid volume gradually increased from <1mL at Day 36 to 950mL at Day 103 (R(2)=0.9275) and amniotic compartment dimensions (length, R(2)=0.9713; width, R(2)=0.9802) increased predictably with fetal growth. Conversely, allantoic fluid protein concentration and volume correlated weakly with fetal age. A significant linear correlation existed between fetal crown rump length (CRL) and crown nose length (R(2)=0.9899) confirming that either measurement can be employed in the ultrasonographic estimation of fetal age. The amniotic compartment and fetometric data presented here have both research and clinical value, particularly in relation to fetal development evaluation and pregnancy viability diagnosis.

Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation.

Although methods to assess testis cell populations are established in mice, the detailed validation of similar methods for bovine testis cells is necessary for the development of emerging technologies such as male germ cell transplantation. As young calves provide donor cells for germ cell transplantation, we characterized cell populations from three key pre-pubertal stages. Nine Angus bull calves were selected to represent three stages of testis development at ages (and testis weights) of 2-3 months (Stage 1, 10 g), 4-5 months (Stage 2, 35 g), and 6-7 months (Stage 3, 70 g). The proportion and absolute numbers of germ and somatic cells in fixed sections and from enzymatically dissociated seminiferous tubules were assessed. Germ cells were identified by DBA and PGP9.5 staining, and Sertoli cells by vimentin and GATA-4 staining. Staining of serial sections confirmed that DBA and PGP9.5 identified similar cells, which were complementary to those stained for vimentin and GATA-4. In fixed tubules, the proportion of cells within tubules that were positive for DBA and PGP9.5 increased nearly three-fold from Stage 1 to Stage 2 with no further increase at Stage 3. Absolute numbers of spermatogonia also increased between Stages 1 and 2. After enzymatic dissociation of tubules, three times more DBA- and PGP9.5-positive cells were isolated from Stage 3 testes than from either Stage 1 or 2 testes. A higher proportion of spermatogonia was observed after enzymatic isolation than were present in seminiferous tubules. These data should help to predict the yield and expected proportions of spermatogonia from three distinct stages of testis development in pre-pubertal bull calves.

Successful transplantation of bovine testicular cells to heterologous recipients.

While heterologous germ cell transplantation was successful in pigs and goats, autologous transplantation alone has been reported to result in donor-derived spermatogenesis in cattle. The objective of this study was to investigate whether the transplantation of heterologous germ cells could result in colonization of recipient testes in cattle of different breeds. Testicular cells were isolated from 8 Bos taurus donor bull calves and then transferred into 15 Bos indicus-cross bull calves. All animals were prepubertal, donors were aged 5-7 months and recipients 5-11 months, and scrotal circumferences ranged from 15 to 22 cm. Single cell suspensions of donor testicular cells, prepared by enzymatic digestion, were labelled with fluorescent dyes PKH26 or CFDA-SE, before transfer into the rete testis of recipients under ultrasonographic guidance. To assess the longevity of colonization by donor cells, recipients were castrated 2-30 weeks after cell transfer. Donor cells were observed in 15/25 (60%) of the testes that received PKH26-labelled cells, whereas no CFDA-SE-positive cell was identified in any recipients. The maturity of the donors or recipients (measured by scrotal circumference) did not affect colonization potential. In freshly isolated tubules, clumps of PKH26-positive cells were observed, which indicated either cell division or extensive local colonization of specific areas of the tubules. In frozen sections, PKH26-positive cells were identified on the seminiferous tubule basement membrane, which indicated that these cells had successfully migrated from the tubule lumen and were likely to be spermatogonia. We conclude that PKH26 was more suitable for labelling donor testis cells and donor cells can be identified up to 6 months following transfer. These results indicate that allogeneic transplantation of testicular cells can occur between Bos taurus and Bos indicus cattle. Further studies will investigate functionality of transferred testicular cells.

Abnormal expression of trophoblast major histocompatibility complex class I antigens in cloned bovine pregnancies is associated with a pronounced endometrial lymphocytic response.

Early embryonic losses are much higher in nuclear transfer (cloned) pregnancies, and this is a major impediment to improving the efficiency of cloned animal production. In cattle, many of these losses occur around the time of placental attachment from the fourth week of gestation. We studied the potential for altered immunologic status of cloned pregnancies to be a contributing factor to these embryonic losses. Expression of major histocompatibility complex class I (MHC-I) by trophoblast cells and distribution of endometrial T-lymphocyte numbers were investigated. Six 5-wk-old cloned pregnancies were generated, and 2 others at 7 and 9 wk were also included, all derived from the same fetal cell line. All 8 cloned placentas displayed trophoblast MHC-I expression. None of the 8 controls (4-7 wk old) showed any MHC-I expression. The percentage of trophoblast cells expressing MHC-I varied in the clones from 17.9% to 56.5%. Numbers of T lymphocytes (CD3(+) lymphocytes) were significantly higher in the endometrium of the majority of cloned pregnancies compared with controls. In the cloned pregnancies, large aggregates of T cells were frequently observed in the endometrium in addition to increased numbers of diffusely spread subepithelial lymphocytes. As trophoblast MHC-I expression is normally suppressed during early gestation, the observed MHC-I expression in the cloned pregnancies is likely to have induced a maternal lymphocytic response that would be detrimental to maintaining viability of the cloned pregnancy. These findings support a role for immunologic rejection in the syndrome of early embryonic loss in cloned bovine pregnancies.