ART Audits Australasia, Northbridge, New South Wales, Australia

SUMMARY
Accreditation of laboratories who perform diagnostic semen analysis in Australia and New Zealand is a requirement of the healthcare system. Within the accreditation process laboratories are required to set ISO standards within their policies and procedures. In order to achieve their aims, laboratories need to be able to measure a number of defined semen parameters both accurately and repetitively, especially around the lower limit of the reference intervals. The methods documented in the WHO-manual are used almost universal as the laboratory standard. Some laboratories incorporate minor method variations into their procedures. As part of the ISO requirements all variations require validation using internally approved processes that are documented and that incorporate appropriate statistical analysis and comparison of results. Validation is an ongoing process and regular review is essential. Evidence of the validation must be available for review by external auditors during accreditation. Where any validated variant method returns results that are significantly different to any method within the WHO-manual, the laboratory needs to develop its own, in-house reference interval for that method.

Reproductive Biology 2011 Suppl. 3: 5-15.

¹ Address for correspondence: PO Box 729, Joondalup, WA 6919, Australia; e-mail: ipik8058@bigpond.net.au

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School of Veterinary Studies, Murdoch University, Murdoch, Australia

SUMMARY
This article traces the history of the work on mammalian embryos carried out by the author, his students and related scientists. It traces the work from the initial experiments at the King Ranch Laboratories, University of Pennsylvania in 1966, the set up of an embryo culture laboratory at the Department of Veterinary Physiology, University of Sydney and the work within that laboratory from 1967 to 1974. This is followed by an account of the author’s subsequent work at Murdoch University from 1975 till his retirement in 1996. The significant role of some of the author’s graduate students in human IVF is also documented.

Reproductive Biology 2011 Suppl. 3: 17-29.

¹ Address for correspondence: Professor Emeritus, School of Veterinary and Biomedical Sciences, Murdoch University, 90 South Street, Murdoch, Western Australia 6150, Australia; e-mail: ray.wales@bigpond.com

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PIVET Medical Centre, Perth, Western Australia, and Cairns Fertility Centre, Cairns, Queensland, Australia

SUMMARY
This invited presentation is intended to cover clinical developments in the evolution of assisted reproductive technology (ART), a process which was attempted during the 1940’s and 50’s and culminated in the first fruition in 1978. The first in vitro fertilisation (IVF) child ensued following the partnership by a scientist with a focussed ambition (Nobel laureate Robert Edwards) joining with the gynaecologist who introduced laparoscopy to Britain in the late 60’s (Patrick Steptoe). My journey commenced in 1976 as a clinician who became immersed in the embryological and endocrinological science, whence most progress in ART emanates, and continued into a medical directorship position from which this personal view is documented. Several clinical advances have been important developments in the understanding and management of sub-fertile patients. However evolution of the various laboratory sciences has been the major key essential to meeting both the immediate as well as the long-term needs for human reproduction. The future requires a much better understanding and control over gametogenesis and a laboratory process which much more closely duplicates intrinsic reproductive physiology, avoiding gamete and embryo exposure to the atmosphere.

Reproductive Biology 2011 Suppl. 3:31-42.

¹ Address for correspondence: Pivet Medical Centre, 166-168 Cambridge Street, Leederville WA 6007, Australia; e-mail: jlyovich@pivet.com.au

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² School of Animal Biology (M085), Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, Australia;

³ UWA Institute of Agriculture (M082), The University of Western Australia, Crawley, Australia

SUMMARY
For the emu, where monogamous mating is normal, artificial insemination (AI) promises much faster genetic improvement and a considerable reduction in production costs by reducing the number of male birds needed for mating. Semen collection is now a routine procedure so the next step is to develop successful protocols for sperm storage. In this paper, we briefly overview our recent progress on the development of protocols for liquid storage and cryopreservation of emu spermatozoa. We have shown that emu semen can be stored at 10°C for up to 48 h with a minimal loss of viability, and that cryopreservation with dimethylacetamide (DMA) as a cryoprotectant is feasible because we have observed no adverse effects of this cryoprotectant on the emu sperm membrane integrity, morphology and motility. We now need to establish the predictability of the various tests in vivo, but the proportions of live normal and motile sperm with good egg membrane penetration potential suggest that acceptable numbers of competent sperm are preserved and that this will be sufficient for AI.

Reproductive Biology 2011 Suppl. 3: 43-49.

¹ Corresponding author: School of Animal Biology M085, Faculty of Natural & Agricultural Sciences, The University of Western Australia, Crawley, Australia. e-mail: irek.malecki@uwa.edu.au

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Genea, Sydney, Australia

SUMMARY
Comparative genomic hybridization (CGH) is an indirect DNA-based test which allows for the accurate analysis of aneuploidy involving any of the 24 types of chromosomes present (22 autosomes and the X and Y sex chromosomes). Traditionally, embryos have been screened using fluorescence in situ hybridization (FISH) – a technique that was limited in the number of chromosomes able to be identified in any one sample. Early CGH reports on aneuploidy in preimplantation embryos showed that any of the 24 chromosomes could be involved and so FISH methods were going to be ineffective in screening out abnormal embryos. Our results from routine clinical application of array CGH in preimplantation genetic diagnosis (PGD) patients confirm previous reports on patterns of chromosomal contribution to aneuploidy. The pregnancy outcomes following embryo transfer also indicate that despite the requirement to freeze embryos, rates are encouraging, and successful ongoing pregnancies can be achieved.

Reproductive Biology 2011 Suppl. 3: 51-60.

¹ Corresponding author: Genea, Level 2, 321 Kent Street, Sydney NSW 2000, Australia; e-mail: maria.traversa@genea.com.au

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School of Animal Biology M085, Faculty of Natural & Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia

SUMMARY
Several factors such as season, genetics, social interaction and metabolic status control or modulate the reproductive capacity of sheep. In addition to these well-studied factors in sheep, the influence of emotional reactivity on the reproductive success of sheep has started to be investigated over the last two decades. In this paper, after briefly reviewing the impact of classical factors affecting reproduction in sheep, we define emotional reactivity and the expression of its inter-individual variability, named temperament. Then, following a description of the protocol to measure temperament in sheep and discussion on the heritability of temperament traits, we illustrate how this selection affects the reproductive biology of sheep. We will be mainly using results obtained from a unique flock of sheep selected for low or high emotional reactivity. In conclusion, we propose that energy partitioning could be one of the mechanisms by which selection for temperament in sheep affects the different steps of the reproductive cycle.

Reproductive Biology 2011 Suppl. 3: 61-77.

¹ Corresponding author: School of Animal Biology M085, Faculty of Natural & Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia; e-mail: Dominique.Blache@uwa.edu.au

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² Institute of Fundamental and Applied Biology, University of Caen Basse-Normandie, Caen;

³ INRA, University of Caen Basse-Normandie, Caen, France

SUMMARY
Aromatase that irreversibly transforms androgens into estrogens is present in the smooth endoplasmic reticulum of nearly all cell types in the mammalian testis. In rodents, all testicular cells except for myoid cells express aromatase activity. We have demonstrated the presence of the functional aromatase (transcript or protein, and biological activity) in adult rat germ cells including pachytene spermatocytes and round spermatids. We have also demonstrated estrogen output from these cells equivalent to that of Leydig cells. Unlike androgen receptors, which are localized mainly in testicular somatic cells, estrogen receptors are present in both somatic and germ cells in the testis. Moreover, we have recently described the rapid membrane effects of estrogens (via G protein-coupled receptor [GPER]) in purified rat germ cells. On the basis of various experimental models, in vitro studies and clinical data, it can be concluded that estrogens play an essential role in male reproduction, specifically in the development of spermatozoa.

Reproductive Biology 2011 11 3: 174-193.

¹ Corresponding author: EA 2608, USC2006 INRA, University of Caen Basse-Normandie, Esplanade de la Paix-14032, CAEN Cedex, France; e-mail: serge.carreau@unicaen.fr

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² Department of Lake and River Fisheries, University of Warmia and Mazury, Olsztyn, Poland;

³ Department of Aquaculture, Szent István University, Gödöllő, Hungary;

⁴ Department of Zoology, University of Warmia and Mazury, Olsztyn, Poland

SUMMARY
To improve controlled reproduction of Eurasian perch Perca fluviatilis, the criteria for the evaluation of final oocyte maturation stages were revised. The new classification covers six preovulatory maturational stages (I -VI) from the end of vitellogenesis to germinal vesicle breakdown (GVBD) and was based on macroscopic changes of preovulatory oocytes (position of the germinal vesicle, GVBD, oil droplets coalescence). The observation was performed during out-of-season artificial reproduction with the use of a single hCG injection (500 IU/kg). The classification was subsequently verified with the controlled reproduction of wild female perch with the use of hormonal stimulation (500 IU hCG/kg of body weight at 12°C). The females were at different maturational stages and constituted respective experimental groups (I-VI). During the experiment, ovulation appeared to be considerably synchronized within particular groups. Statistical differences in latency time (time between hormonal treatment and ovulation) were found between experimental groups (mean latency time: 110, 92, 68, 49, 29 and 18 h in groups representing VI, V, IV, III, II and I stage of the proposed classification, respectively). The proposed classification and the results presented in the study allowed for effective synchronisation of ovulation. The use of our new oocyte maturation classification may positively influence the effectiveness of Eurasian perch production.

Reproductive Biology 2011 11 3: 194-209.

¹ Corresponding author: Department of Lake and River Fisheries, University of Warmia and Mazury, Oczapowskiego 5; 10-957 Olsztyn, Poland; e-mail: danielzarski@interia.pl

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² Department of Animal Physiology, Faculty of Biology, University of Warmia and Mazury in Olsztyn;

³ Department of Public Health and Epidemiology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn;

⁴ MSWiA Hospital in Olsztyn, Department of Oncology;

⁵ Department of Human Physiology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn

SUMMARY
Aquaporin 1 (AQP1) is a water channel protein expressed in endothelial and epithelial cells of many tissues, including the vasculature, where it serves to increase water permeability of the cell membrane. The aim of this study was to investigate the expression and distribution of AQP1 in porcine peri-ovarian vascular complex (PVC) during the estrous cycle and early pregnancy. Immunohistochemistry and semi-quantitative immunoblotting techniques were used. We have demonstrated the presence of AQP1 protein in the endothelial cells of the lymphatic and vascular endothelium of the PVC during the pig estrous cycle and early pregnancy. The expression of AQP1 protein in the PVC did not change significantly between Days 10-12 and 14-16, but increased on Days 2-4 and 18-20 when compared with Days 10-12 and 14-16 of the estrous cycle. In pregnant gilts, the expression of AQP1 did not differ significantly during the onset and the end of the implantation process and also when compared to the mid- and late-luteal phases of the estrous cycle. In conclusion, AQP1 is expressed in the endothelial cells of PVC and may modulate hormonal regulation of reproductive organs.

Reproductive Biology 2011 11 3: 210-223.

¹ Corresponding author: University of Warmia and Mazury in Olsztyn; Department of Animal Physiology; Oczapowskiego 1A; 10-718 Olsztyn, Poland; e-mail: skowron@uwm.edu.pl

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² Poznan University of Life Science, Department of Genetics and Animal Breeding, Poznan, Poland;

³ Veterinary Research Institute, Brno, Czech Republic

SUMMARY
Chromosomal imbalance in gametes and embryos is one of the factors contributing to early embryonic mortality. Although the rate of chromosomally abnormal sperm cells is low and usually does not exceed 1%, there is no clear indication of fertilizing potential of such gametes. The aim of the experiment was to investigate the type and incidence of numerical chromosomal aberrations in spermatozoa produced by fertile boars used in artificial insemination (AI). We used the protocol of fluorescent in situ hybridization (FISH) on sperm interphase nuclei with molecular probes for porcine chromosome pairs 1 and 10. Altogether 12 348 sperm cells were examined. Disomy was observed in spermatozoa of all seven AI boars whereas only one diploid cell was identified in all screened sperm cells. The average rate of chromosomally unbalanced sperm was 0.105% (13/12 348) with an inter-individual variation from 0.048% to 0.194%. Among abnormal sperm cells, both disomy (0.097%) and diploidy (0.008%) were detected. Nullisomy was not included into calculations. The estimated aneuploidy rate calculated by doubling the number of disomic cells was 0.194%. Chromosome pair 10 was significantly more often involved in non-disjunction (75%, 9/12 aneuploid sperm cells) than chromosome pair 1 (25%, 3/12). We have shown for the pig that the rate of disomic cells falls into a range presented by other authors, whereas that of diploid spermatozoa appeared to be lower in the present study. In conclusion, numerical chromosome aberrations were present in spermatozoa of all AI boars analyzed in this study. Therefore, it can be assumed that the presence of unbalanced spermatozoa at the level observed in fertile males does not significantly affect their reproductive potential.

Reproductive Biology 2011 11 3: 224-235.

¹ Corresponding author: Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Wolynska 33, 60-637 Poznan, Poland; e-mail: lechniak@jay.au.poznan.pl

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