Time within reproductive season, but not age or inbreeding coefficient, affects seminal and sperm quality in the whooping crane (Grus americana).

All living whooping cranes (Grus americana) are descended from 16 or fewer birds that remained alive in the early 1940s, a bottleneck that puts the species at potential risk for inbreeding depression. Although AI is commonly used in the management of the captive population of this species, little is known about seminal traits or factors affecting sperm quality in the whooping crane. In the present study, semen samples were collected from 29 adult males (age 3-27 years) during the early (March), mid (April) and late (May) breeding season over 2 consecutive years. The effects of donor age, time within reproductive season and level of inbreeding on seminal characteristics were analysed using regression and information-theoretic model selection. Only time within reproductive season significantly affected seminal traits, with total numbers of spermatozoa and proportions of pleiomorphisms increasing across the season. We conclude that, even with a highly restricted number of founders, there is no discernible influence of inbreeding (at the levels described) on sperm output or quality. Furthermore, although there is variance in seminal quality, the whooping crane produces significant numbers of motile spermatozoa throughout the breeding season, similar to values reported for the greater sandhill crane (Grus canadensis tabida).

Source of protein supplementation during in vitro culture does not affect the quality of resulting blastocysts in the domestic cat.

The objective of this study was to assess and compare the quality of cat blastocysts produced in vitro using commercial blastocyst growth media supplemented with different sources of proteins (serum protein substitute from in vitro maturation through embryo development vs 4 mg/ml of bovine serum albumin for maturation and 5% foetal calf serum for fertilization and embryo development). Impact was specifically examined on the proportion of blastocyst formation, total number of blastomeres, proportion of inner cell mass and expression of pluripotency marker proteins NANOG and OCT-4. Blastocyst formation per total cleaved embryos was similar (p > 0.05) regardless of the protein supplementation. There were no differences (p > 0.05) between culture conditions regarding average number of blastomeres and proportion of inner cell mass in each embryo. Presence of OCT-4 protein was detected in nuclei of both trophectoderm and inner cell mass region, with a stronger signal in the latter regardless of the culture medium. NANOG protein also was present in the inner cell mass regardless of the in vitro culture condition. We therefore demonstrated that serum protein substitute was as good as semi-defined protein sources for the production of good-quality blastocysts and embryonic stem cells. In addition, a single defined medium could be successfully used for cat oocyte maturation, in vitro fertilization and embryo development.

Molecular markers of spermatogonial stem cells in the domestic cat.

Spermatogonial stem cells (SSCs) represent an exciting new avenue for assisted reproduction in endangered and genetically valuable species. Before this technology can be applied to wildlife, species-specific markers are required to evaluate SSC enrichment strategies and monitor subsequent in vitro culture. This study was designed to evaluate six conserved SSC markers (THY1, GPR125, GFRalpha1, PLZF, UCHL1 and OCT4) in the cat. Testes from three juveniles and three adults were obtained following routine castrations and processed for mRNA extraction. RT-PCR of whole testis and cell suspensions enriched for SSCs by differential plating confirmed that all six SSC markers are expressed in both the whole testis and SSC-enriched cell fractions. The expression of all six putative SSC marker genes in the cat testis suggests conservation of SSC markers, and perhaps self-renewal mechanisms, in felids.

Lessons learned from nuclear transfer (cloning).

Somatic cell nuclear transfer (SCNT) has been accomplished in an ever-growing list of species. In each case, an enucleated oocyte has successfully reset the nucleus of a somatic cell such that the embryonic program could progress to the production of a live offspring. The overall efficiency of the process remains low due to a combination of biological and technical challenges, some of which are known and others remain to be elucidated. Comparative studies between livestock and laboratory species may help improve not only nuclear transfer efficiencies but also uncover basic underlying developmental principles.

Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates.

Embryonic stem (ES) cell lines provide an invaluable research tool for genetic engineering, developmental biology and disease models. These cells can be maintained indefinitely in culture and yet maintain competence to produce all the cells within a fetus. While mouse ES cell lines were first established over two decades ago and primate ES cells in the 1990 s, validated ES cell lines have yet to be established in ungulates. Why competent, pluripotent ES cells can be established from certain strains of mice and from primates, and not from cows, sheep, goats or pigs is an on-going topic of interest to animal reproduction scientists. The identification of appropriate stem cell markers, functional cytokine pathways, and key pluripotency-maintaining factors along with the release of more comprehensive bovine and porcine genomes, provide encouragement for establishment of ungulate ES cell lines in the near future.

State of the art in the production of transgenic goats.

This review summarises recent advances in the field of transgenic goats for the purpose of producing recombinant proteins in their milk. Production of transgenic goats via pronuclear microinjection of DNA expression vectors has been the traditional method, but this results in low efficiencies. Somatic cell nuclear transfer has dramatically improved efficiencies in rates of transgenesis. Characterisation of transfected cells in vitro before use in nuclear transfer guarantees that kids born are transgenic and of predetermined gender. Using these platform technologies, several recombinant proteins of commercial interest have been produced, although none of them has yet gained marketing approval. Before these technologies are implemented in goat improvement programmes, efficiencies must be improved, costs reduced, and regulatory approval obtained for the marketing of food products derived from such animals.

Production of bioproducts through the use of transgenic animal models.

Transgenic livestock that produce recombinant proteins in their milk can provide an economic and safe system for production of valuable proteins, such as pharmaceutical proteins for treatment or prevention of human disease or biomaterials for medical use. This method of production is frequently referred to as biopharming. The promise of biopharming, that is the actual commercial production of pharmaceuticals and other bioproducts, is nearing fulfillment. Improvements in molecular and reproductive techniques and strong economic incentives have continued to drive the implementation of transgenic technology to domestic animals. Nuclear transfer using transgenic donor cells is rapidly becoming the predominant technique used in the production of transgenic livestock, replacing the direct injection of DNA into the zygotic pronuclei. Production of transgenic founder animals by nuclear transfer in combination with traditional reproductive technologies can result in the propagation of transgenic herds of sufficient size to meet market demands for commercially important proteins. While some of the companies that have established transgenic programs have run into setbacks owing to a combination of economic, scientific and regulatory difficulties, other companies are continuing to make significant advances. While further improvements are needed to increase efficiencies of production, economically viable production of recombinant proteins using livestock species is not only possible but should be a commercial reality in the very near future.

Prepubertal propagation of transgenic cloned goats by laparoscopic ovum pick-up and in vitro embryo production.

The use of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production was evaluated in the early propagation of cloned goats. Ten kinder goats produced by somatic cell nuclear transfer technology were used as oocyte donors. Half of the donor animals were subjected to LOPU at 2-3 months of age (prior to induction of lactation), whereas the other five goats were subjected to LOPU at 6-7 months of age (following induction to lactation). They were stimulated with 80 mg NIH-FSH-P1 (Folltropin, Vetrepharm, Canada) together with 300 IU eCG (Novormon, Vetrepharm, Canada) administered intramuscularly 36 h prior to LOPU. The number of follicles aspirated and oocytes recovered was higher in the younger group of donors (57 +/- 7 and 41 +/- 4 vs. 28 +/- 2 and 25.8 +/- 2, p < 0.05), however, oocytes from animals in the late prepubertal age showed higher developmental capacity resulting in higher transferable embryo yield (81.4% vs. 67.8%, p < 0.01), pregnancy rate (80% vs. 40%, p < 0.05) and total kids born (27 vs. 15, p < 0.01). In conclusion, LOPU in combination with in vitro embryo production techniques is an efficient method for the early propagation of valuable goats produced by somatic cell nuclear transfer.

Transgenic goats produced by DNA pronuclear microinjection of in vitro derived zygotes.

This study was undertaken to investigate various factors affecting the outcomes of in vitro fertilization (IVF) of oocytes retrieved by laparoscopic ovum pick-up (LOPU) technique from prepubertal and adult goats, as well as to evaluate the developmental competence of in vitro produced embryos. Oocyte-cumulus complexes recovered by LOPU from donors stimulated with gonadotrophins were matured in vitro. Fresh semen was used for IVF following various capacitation treatments. In vitro produced zygotes were either cultured to assess in vitro development or were transferred into recipients for full term development. The results indicated that successful IVF of the goat oocytes was affected by factors such as sperm capacitation treatment, oocyte quality, and abundance of cumulus cells on zona pellucida. Oocytes from both prepubertal and adult goats demonstrated similar full term developmental competence despite the fact that in vitro developmental rates were lower for prepubertal goats. The births of transgenic offspring demonstrated that the established LOPU-IVF technology combined with pronuclear microinjection can be successfully used to produce transgenic goats.

Advances in the production and propagation of transgenic goats using laparoscopic ovum pick-up and in vitro embryo production technologies.

Laparoscopic ovum pick-up (LOPU) is a convenient methodology by which oocytes can be recovered and used either for in vitro production of zygotes or as a source of cytoplasts in nuclear transfer (NT) procedures. The pregnancy and transgenesis rates achieved with IVM/IVF of LOPU-sourced oocytes followed by subsequent DNA microinjection of zygotes are similar to the rates obtained when using in vivo-produced oocytes or zygotes. Similarly, pregnancy rates and kids born by using LOPU-sourced and in vitro matured oocytes as recipient cytoplasts in NT programs are comparable with those reported by others using in vivo matured oocytes collected by oviduct flushing. The use of LOPU allows for improved control over the stage of maturation/development of the oocytes and produced zygotes, a less invasive means of recovery, thereby allowing for repeated usage of the oocyte donor animals and the ability to source the oocytes from live animals of known health status. In addition, because of large follicular responses that can be obtained from prepubertal animals, LOPU followed by IVM/IVF has demonstrated great potential for the early propagation of valuable animals, in particular, transgenic animals.

Production of cloned goats after nuclear transfer using adult somatic cells.

The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.

Seasonal variation in preovulatory events associated with synchronization of estrus in dwarf goats.

This experiment was conducted to define the temporal relationships among estrus, the LH surge and ovulation after estrus synchronization in dwarf goats and to assess the effect of season on these parameters. In November (breeding season), March (transition period) and July (non-breeding season), estrus was synchronized in 12 dwarf goats by means of intravaginal sponges containing 60 mg medroxyprogesterone acetate (MAP) for 10 d, coupled with 125 microg cloprostenol i.m. 48 h before sponge removal and 300 IU eCG i.m. at sponge removal. A different group of animals was used during each time period. Onset of estrus was monitored using two males, and blood samples for the measurement of plasma LH were collected at 2-h intervals from 24 to 60 h after sponge removal. Ovulation was confirmed by laparoscopy at 54 and 72 h after sponge removal. A seasonal shift was detected in the intervals to onset of estrus, LH surge, and ovulation after sponge removal (P<0.05), with sponge removal to onset of estrus being shorter (P<0.05) in November (25.0 +/- 1.56 h) and July (28.9 +/- 2.43 h) than in March (40.9 +/- 3.27 h). The intervals between onset of estrus and the LH surge and between the LH surge and ovulation were found to be constant throughout the different seasons. An optimal time for breeding, artificial insemination, oocyte and embryo recovery, and embryo transfer may be predicted using information gained from these studies.

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes.

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

The infrared spectroscopic Gram stain.

The attenuated total reflectance (ATR) spectra of bacterial colonies were obtained in situ, without their removal from the agar growth media using the infrared microscope. Principal components regression (PCR) analysis was used to obtain a measure of the differentiating potential of the ATR spectra obtained on two standard sets consisting of 31 and 44 species, respectively, grown on trypticase soy agar. A quantitative response value of +1 was assigned to Gram positive species and -1 was assigned to Gram negative species. In a cross validation experiment, the Gram stain property correlated with the factor loadings with a multiple correlation of 0.9502 and 0.9520 for each of the standard sets, respectively. Also, it was found that ATR spectra can be obtained from bacteria grown on blood agar and still have good differentiating capabilities for Gram stain. In addition, ATR spectra provide significantly better differentiating spectral information and predicting capabilities than specular reflectance spectra obtained in situ. Finally, the numerical value obtained for the Gram stain can be used as a measure of Gram variability. Results indicate that the Gram positive values shift to Gram negative values as the bacterial cell wall deteriorates with time.

Sexual maturation and fertility of male Nigerian Dwarf goat (Capra hircus) clones produced by somatic cell nuclear transfer.

Three, genetically identical, Nigerian Dwarf bucks produced by somatic cell nuclear transfer (NT) of fetal fibroblasts were monitored for sexual maturation and fertility. Starting at four months of age, these male clones were trained to serve an artificial vagina (AV). Average age of the NT-derived bucks at first semen collection was 20 weeks, which was not different from that of other young bucks of this breed (average age at first collection = 20 weeks). Average sperm production at 5 months of age for the NT-derived bucks was 5.0 x 10(8) spermatozoa, which was comparable to that of dwarf bucks of similar age (3.4 x 10(8) spermatozoa). At seven months of age, semen collected from two NT-derived bucks was used to artificially inseminate six females (three does per buck). Five does were confirmed pregnant by ultrasound at day 42. Nine healthy kids, four males and five females, were born in March and April 2000. Viable spermatozoa were collected from one of the F1 males at 28 weeks of age. These results demonstrated that NT-derived bucks and one of their male offspring developed sexually within the normal timeframe for their breed and that the clones were fertile.

Reovirus-induced G(2)/M cell cycle arrest requires sigma1s and occurs in the absence of apoptosis.

Serotype-specific differences in the capacity of reovirus strains to inhibit proliferation of murine L929 cells correlate with the capacity to induce apoptosis. The prototype serotype 3 reovirus strains Abney (T3A) and Dearing (T3D) inhibit cellular proliferation and induce apoptosis to a greater extent than the prototype serotype 1 reovirus strain Lang (T1L). We now show that reovirus-induced inhibition of cellular proliferation results from a G(2)/M cell cycle arrest. Using T1L x T3D reassortant viruses, we found that strain-specific differences in the capacity to induce G(2)/M arrest, like the differences in the capacity to induce apoptosis, are determined by the viral S1 gene. The S1 gene is bicistronic, encoding the viral attachment protein sigma1 and the nonstructural protein sigma1s. A sigma1s-deficient reovirus strain, T3C84-MA, fails to induce G(2)/M arrest, yet retains the capacity to induce apoptosis, indicating that sigma1s is required for reovirus-induced G(2)/M arrest. Expression of sigma1s in C127 cells increases the percentage of cells in the G(2)/M phase of the cell cycle, supporting a role for this protein in reovirus-induced G(2)/M arrest. Inhibition of reovirus-induced apoptosis failed to prevent virus-induced G(2)/M arrest, indicating that G(2)/M arrest is not the result of apoptosis related DNA damage and suggests that these two processes occur through distinct pathways.

Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI).

In the human and the mouse, intracytoplasmic sperm injection (ICSI) apparently triggers normal fertilization and may result in offspring. In the bovine, injection of spermatozoa must be accompanied by artificial methods of oocyte activation in order to achieve normal fertilization events (e.g., pronuclear formation). In this study, different methods of oocyte activation were tested following ICSI of in vitro-matured bovine oocytes. Bovine oocytes were centrifuged to facilitate sperm injection, and spermatozoa were pretreated with 5 mM dithiothreitol (DTT) to promote decondensation. Sperm-injected or sham-injected oocytes were activated with 5 microM ionomycin (A23187). Three hours after activation, oocytes with second polar bodies were selected and treated with 1.9 mM 6-dimethylaminopurine (DMAP). The cleavage rate of sperm-injected oocytes treated with ionomycin and DMAP was higher than with ionomycin alone (62 vs 27%, P < or = 0.05). Blastocysts (2 of 41 cleaved) were obtained only from the sperm-injected, ionomycin + DMAP-treated oocytes. Upon examination 16 h after ICSI, pronuclear formation was observed in 33 of 47 (70%) DMAP-treated oocytes. Two pronuclei were present in 18 of 33 (55%), while 1 and 3 pronuclei were seen in 8 of 33 (24%) and 7 of 33 (21%) oocytes, respectively. In sham-injected oocytes, pronuclear formation was observed in 15 of 38 (39%) with 9 (60%) having 2 pronuclei. Asa single calcium stimulation was insufficient and DMAP treatment could result in triploidy, activation by multiple calcium stimulations was tested. Three calcium stimulations (5 microM ionomycin) were given at 30-min intervals following ICSI. Two pronuclei were found in 12 of 41 (29%) injected oocytes. Increasing the concentration of ionomycin from 5 to 50 microM resulted in a higher rate of activation (41 vs 26%). The rate of metaphase III arrest was lower while the rate of pronuclear formation and cleavage development was higher in sperm-injected than sham-injected oocytes, suggesting that spermatozoa contribute to the activation process. Further improvements in oocyte activation following ICSI in the bovine are necessary.

Neer Award 1999. Overuse activity injures the supraspinatus tendon in an animal model: a histologic and biomechanical study.

Overuse activity has been implicated as an etiologic factor in injury to the rotator cuff and to the supraspinatus tendon in particular. Due in part to the lack of an appropriate animal model, expex85ental studies have not addressed this issue. With the use of a rat model, we measured the effects of an overuse running regimen on 36 Sprague-Dawley rats after 4 (n = 12), 8 (n = 12), or 16 (n = 12) weeks of exercise and compared them with a control group of rats (n = 10) who were allowed normal cage activity. The histologic characteristics, the gross morphologic characteristics, and the mechanical properties of the tendon tissue were evaluated. The supraspinatus tendons in the exercised animals demonstrated significant changes as a result of overuse at all time points compared with the normal group. There was an increase in cellularity and a loss of the normal collagen fiber organization consistent with what has been seen in human tendinopathy. The tendons from the exercise groups were larger than normal in cross-sectional analysis at 4 weeks (129% of control, P < .01) and continued to increase in size with time to 16 weeks (164% of control, P = .01). The mechanical properties of the tendons deteriorated in response to overuse exercise with a decreased modulus of elasticity ranging from 52% to 61% of control (P = .07 at 4 weeks, P < .05 at 8 and 16 weeks) and a decreased maximum stress of failure ranging from 51% to 63% of control (P < .007). These findings support overuse activity as an etiologic factor in the development of supraspinatus tendinopathy and begin to describe the changes in the tendons as a result of such activity. This model can now be used to study the effect of various treatment modalities on these injuries.