Application Of Assisted Reproductive Technologies Research Articles

Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos

The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively imprinted genes ( Ata3, Dlk1, Gnas, Grb10, Magel2, Mest- 1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos. The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest- 1 showed differential expression in Day 21 parthenogenetic embryos compared to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation.

Ultrasonographic-guided retrieval and in vitro maturation of eland ( Taurotragus oryx) and bongo ( Tragelaphus eurycerus isaaci) antelope oocytes

The limited availability of gametes is a major factor hindering the development and application of assisted reproductive technologies (ART) in large non-domestic ungulates. This is partly due to the small number of captive animals and handling difficulties associated with procedures for gamete recovery. In the present study, results are reported of multi-year studies on ovarian stimulation and oocyte retrieval by ultrasonographic-guided transvaginal follicular aspiration and subsequent in vitro maturation (IVM) in eland and bongo antelopes. All procedures were conducted on sedated females handled in a hydraulic chute without inducing general anesthesia. Five estrous synchronization/ovarian stimulation protocols were evaluated and data are presented on 73 and 15 procedures in eland and bongo, respectively. Repeating procedures (≤once/month) on the same female did not affect ovarian response or number oocytes recovered in either species. Eland females, but not the ovarian stimulation treatment, affected ovarian response. Ovarian stimulation treatment affected oocyte recovery rate in eland, but not in bongo. In both species, ovarian hormone stimulation treatment affected the distribution of follicles by size and the status of expansion of the cumulus cell investment of oocytes, but not the frequency of metaphase II oocytes during IVM. The timing of extrusion of the first polar body during IVM was more synchronous in bongo than in eland oocytes. It is concluded that Transvaginal oocyte retrieval (TVOR) can be safely and repeatedly applied in gonadotropin-treated eland and bongo females to recover oocytes that can mature in vitro. The methods described for the present study can be adapted to improve the availability of non-domestic ungulate oocytes for basic and applied studies.

Ultrasound examination and behavior scoring of captive broadnose sevengill sharks, Notorynchus cepedianus (Peron, 1807)

Serial ultrasound examination of four mature female sevengill sharks (Notorynchus cepedianus) was carried out over 18 months. Monitoring the reproductive cycle and development of follicles and fetuses in sharks in a noninvasive manner using this technique has not been reported previously. Sharks were caught out of the "Oceanarium" tank by divers using a specially made catch-out bag, and brought to a holding area for examination. A behavior scoring system was used to monitor the impact of regular handling on the well-being of the animals. Ultrasound showed the growth and regression of follicles in sevengill ovaries, and allowed an approximation of the reproductive stage of these sharks. Monitoring behavior at five time points during the procedure showed that regular handling of sharks for clinical studies could be done with minimal impact on animal welfare. The ability to follow reproductive events in elasmobranches using ultrasonography is an important step in the application of assisted reproductive technology in these species. Assisted reproductive technology, such as monitoring female reproductive cycles and artificial insemination, could potentially be used to maintain genetic diversity and compliment aquaria-based breeding programs for endangered species such as the gray nurse shark (Carcharias taurus). Zoo Biol 26:383-395, 2007. (c) 2007 Wiley-Liss, Inc.

Technologies for fixed-time artificial insemination and their influence on reproductive performance of <I>Bos indicus</I> cattle

The adaptation of Bos indicus cattle to tropical and subtropical environments has led to their widespread distribution around the world. Although artificial insemination (AI) is one of the best alternatives to introduce new genetics into Bos indicus herds, the peculiarity of their temperament and the tendency to show short oestrus (many of them during the night) greatly affects the effectiveness of genetic improvement programs. Therefore, the most useful alternative to increase the number of females that are inseminated is the use of protocols that allow for AI without the need for oestrus detection, usually called fixed-time AI (FTAI). Besides, the development of protocols to advance the resumption of cyclicity during the early postpartum period has a great impact on beef production and will allow for the inclusion of a significantly larger population of animals into genetic improvement programs. Fixed-time AI protocols using progestin devices, oestradiol and eCG have resulted in consistent pregnancy rates in suckled Bos indicus and Bos indicus x Bos taurus cows. Furthermore, fertility in the successive cycles and the overall pregnancy rates at the end of the breeding season, have been shown to be improved by the use of progestin devices at the beginning of the breeding season. In summary, exogenous control of luteal and follicular development has facilitated the application of assisted reproductive technologies in Bos indicus-influenced cattle, by offering the possibility of planning programs without the necessity of oestrus detection and may provide the opportunity to improve reproductive performance of beef cattle in tropical climates.

The temporal relationship between oocyte maturation and early fertilisation events in relation to the pre-ovulatory LH peak and preimplantation embryo development in red deer ( Cervus elaphus)

The temporal relationships among oocyte maturation, gamete transport and fertilisation following the pre-ovulatory luteinsing hormone surge in red deer were established; and secondly, early preimplantation development to the blastocyst stage in relation to the onset of oestrus was determined for red deer. In the first series of observations, oestrus was synchronised in April ( N = 22), for the fixed time recovery of gametes from 0 to 36 h after the estimated pre-ovulatory LH peak. Matings were observed and the time of the LH peak was determined from the retrospective analysis of blood plasma collected at 3 h intervals. Gametes were recovered surgically and the meiotic status of follicular and ovulated oocytes assessed. Spermatozoa were recovered from the oviduct and their motility analysed by videomicroscopy. Nineteen of 22 hinds exhibited a pre-ovulatory LH surge and were observed to mate. Oocyte metaphase I occurred between 11 and 18 h, and metaphase II was completed within the follicle between 20 and 25 h following the pre-ovulatory LH peak. Fertilised ova were recovered from 30 to 36 h in both the ampulla and isthmic portions of the oviduct. Motile spermatozoa were first recovered from the isthmus and the ampulla at 13 and 21 h, respectively, after the LH peak. Hyperactive spermatozoa were observed in both the isthmus and the ampulla flushings but only from the eight hinds that had ovulated. In the second series of observations, 16 mature hinds were synchronised and allocated to groups for embryo collection on days 3, 5 and 7 after oestrus. Eight embryos were recovered; an 8-cell at 90 h, 3 morulae at 137, 138 and 186 h, and 4 blastocysts at 180, 182 and 190 h post-mating. Blastocysts were only recovered from the uterine horns and the mean ± S.E.M. number of nuclei per blastocyst was 93.5 ± 10.0 with a range of 66–114 cells. The results of this study will improve the application of assisted reproductive technologies to red deer as they indicate that oocyte maturation, fertilisation and early embryonic development of the red deer is similar to other domestic ruminants with the exception that the red deer embryo enters the uterus at the blastocyst stage.

The Application of Reproductive Technologies to Natural Populations of Red Deer

Over the past decade, there has been increasing interest in the application of reproductive technology to the conservation and management of natural populations of deer. The application of assisted reproduction technologies within natural population of deer is in its infancy. However, its future potential is enormous, particularly in relation to genetic management or conservation. This paper reviews the present state of such technologies for a wild subspecies of red deer, the Iberian red deer (Cervus elaphus hispanicus), by discussing the major components of oestrous synchronization, semen collection/cryopreservation and insemination techniques. In addition, findings made during the course of studies on natural populations have enormous potential for the understanding of novel reproductive mechanism that may not be uncovered by livestock or human studies. A summary of these results are also reviewed here.

Molecular regulation of blastocyst formation

Preimplantation development encompasses the interval from insemination until embryo implantation and thus includes the ‘freeliving’ period of oviduct and uterine development. Formation of the blastocyst is required for implantation and establishment of pregnancy, and is a principal determinant of embryo quality prior to embryo transfer. Development through this period is regulated by the expression of specific gene families that encode for cell polarity, cell junctional, cytoskeletal, ion transporter, and water channel gene products that direct the acquisition of cell polarity and differentiation of the outer cells of the early embryo. This results in the formation of the trophectoderm, which is the first epithelium of development. This review considers the roles of each of these gene families in trophectoderm differentiation and blastocyst formation. The principal hypothesis under investigation is that blastocyst formation is regulated by a Na/K-ATPase-generated trans-trophectoderm ion gradient that promotes the accumulation of water across the epithelium. This, combined with the formation of the tight junction seal controlling paracellular movement of water between adjacent trophectoderm cells, results in the formation of a fluid-filled blastocyst cavity and the expansion of the blastocyst. Results from recent experiments, however, have cast some doubt on the role of Na/K-ATPase in mediating these events and have defined water channels or Aquaporins (AQPs) as physiological mediators of fluid movement across the trophectoderm. In addition, studies have now implicated mitogen-activated protein kinase (MAPK) signaling as an important mediator of development to the blastocyst stage. Such studies define the physiology of blastocyst formation and serve to support the application of assisted reproductive technologies (ART) to both human and animal species.

Molecular regulation of blastocyst formation

Preimplantation development encompasses the interval from insemination until embryo implantation and thus includes the ‘freeliving’ period of oviduct and uterine development. Formation of the blastocyst is required for implantation and establishment of pregnancy, and is a principal determinant of embryo quality prior to embryo transfer. Development through this period is regulated by the expression of specific gene families that encode for cell polarity, cell junctional, cytoskeletal, ion transporter, and water channel gene products that direct the acquisition of cell polarity and differentiation of the outer cells of the early embryo. This results in the formation of the trophectoderm, which is the first epithelium of development. This review considers the roles of each of these gene families in trophectoderm differentiation and blastocyst formation. The principal hypothesis under investigation is that blastocyst formation is regulated by a Na/K-ATPase-generated trans-trophectoderm ion gradient that promotes the accumulation of water across the epithelium. This, combined with the formation of the tight junction seal controlling paracellular movement of water between adjacent trophectoderm cells, results in the formation of a fluid-filled blastocyst cavity and the expansion of the blastocyst. Results from recent experiments, however, have cast some doubt on the role of Na/K-ATPase in mediating these events and have defined water channels or Aquaporins (AQPs) as physiological mediators of fluid movement across the trophectoderm. In addition, studies have now implicated mitogen-activated protein kinase (MAPK) signaling as an important mediator of development to the blastocyst stage. Such studies define the physiology of blastocyst formation and serve to support the application of assisted reproductive technologies (ART) to both human and animal species.

Life-table analysis of artificial insemination pregnancy rates for couples with male factor and idiopathic infertility.

Background: In the summer of 2002, standard guidelines for the application of assisted reproductive technology were reported by a research group of the Ministry of Health, Labor and Welfare. The present study aimed to examine the relationship between the number of cycles of artificial insemination and the cumulative pregnancy rates according to the cause of infertility. Methods: Patients who experienced their first cycle of artificial insemination during the period of January 1999-December 2002 were included in the study and were divided into a male factor infertility group and an idiopathic infertility group. Cumulative pregnancy rates resulting from artificial insemination with the husband's semen were calculated by the life-table approach. Results: During the study period, 139 couples entered the assisted reproduction program and underwent 581 cycles. Significant differences were observed in cumulative pregnancy rates between the two groups. Conclusion: It is recommended that couples with male factor infertility and who fail to conceive within six or seven cycles of intrauterine insemination, consider a modification of treatment strategy such as in vitro fertilization, because cumulative pregnancy rates of this group were reached at a plateau within six or seven cycles. In contrast, patients with idiopathic infertility, the cumulative pregnancy rates appeared to increase constantly with each subsequent cycle. It is important to consider modifications of treatment strategy in the light of the cause of infertility. (Reprod Med Biol 2004; 3: 27-31).

339CORRELATION BETWEEN FOLLICLE SIZE AND QUALITY OF OOCYTES FROM THE SUPEROVULATED CYNOMOLGUS MONKEY

Variability in the superovulatory response continues to be one of the most frustrating problems with the application of assisted reproductive technologies in non-human primates. Superstimulation of donor animals with equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) is widely used, but individual responses to these hormones vary widely. In human in vitro fertilization, follicle size is commonly used as a marker to determine the timing of hCG treatment in order to acquire oocytes matured in vivo. Incorporation of techniques designed to control follicular size in humans may improve superstimulatory response in non-human primates. In this study, we measured follicle size and examined oocyte quality in Cynomologus Monkeys superstimulated as described below. On the first day of spontaneous menses, monkeys were treated with long-acting GnRH (Luprin: Takeda Pharm, Co. Ltd., Osaka, Japan;; Day 0). A dose of 25IU/kg/day eCG (Serotoropin;; Teikokuzoki Pharm, Co. Ltd., Tokyo, Japan) was administered i.m. from Day 1 to Day 9, followed by 200IUkg−1 hCG (Puberogen;; Sankyo Pharm, Co. Ltd., Tokyo, Japan) i.m. on Day 10. Dominant follicle sizes were measured on Days 7 and 9 by ultrasonography. Oocytes were collected by laparoscopy in anesthetized monkeys 40h after the hCG injection. Oocytes were classified by nuclear status. Immature oocytes at the stages of germinal vesicles (GV) and metaphase (MI) were cultured until reaching the stage of Metaphase II (MII). Matured oocytes (MII) were fertilized by ICSI and cultured for 7 days. At the end of culture, the developmental stage of oocytes was examined. The ovaries with different-sized follicles on Day 7 were divided into two groups;; ovaries with large follicles (>4.5mm) were in the first group and ovaries with small follicles (<4.0mm) were in the second group. On Day 9, follicles in first group grew to more than 5.0mm and follicles in second group remained less than 5.0mm. Sixty-two percent of oocytes from follicles in first group were at MI or MII stage, while only 15% of oocytes in second group reached the MI or MII stage. After ICSI, 42% of MII oocytes from first group developed to the blastocyst stage, while no blastocyts were observed in second group. These results suggest that the size of dominant follicle was a limiting factor for the developmental ability of oocytes in vitro. For production of Cynomolgus monkey blastocysts derived from ICSI, the diameter of dominant follicle was required to be at least 5mm before hCG in order to collect MI and MII oocytes. Incorporation of hormonal treatments designed to optimize follicular size probably reduced the variability in quality of oocytes. Therefore, we expect that an adjustment of dose and duration of eCG and hCG treatment may improve developmental ability of oocytes from follicles that had not reached 5mm.

Evolving ethics in medically assisted reproduction.

Ethical problems arising from the application of assisted reproductive technology are discussed for four specific areas, namely embryo research, multiple pregnancies, preimplantation genetic diagnosis (PGD) for social sexing, and finally PGD with HLA typing.

Pattern and manipulation of follicular development in Bos indicus cattle

Bos indicus cattle are widespread in tropical regions due to their adaptation to these environments. Although data on reproductive performance have indicated both inferior and superior results for B. indicus cattle, there is little doubt that B. indicus cattle are superior than Bos taurus cattle when they are both kept in tropical or subtropical environments, where stressors like hot temperatures, humidity, ectoparasites and low quality forages are greater. Reproductive endocrinology and oestrus behaviour of the B. indicus cattle have been studied for over 30 years; however, the application of technologies such as real time ultrasonography and Heat-Watch systems has expanded our knowledge on the ovarian follicular-wave dynamics during the oestrous cycle and the time of ovulation. Ovarian follicular dynamics in B. indicus cattle is characterised by the occurrence of two, three or sometimes four waves of follicular development. While dominance is similar to that in B. taurus cattle, maximum diameters of the dominant follicle and CL are smaller than those reported in B. taurus and are probably due to a lower capacity for LH secretion than in B. taurus. Duration of oestrus is approximately 10 h and the interval from oestrus to ovulation is about 27 h. However, the variability in response to prostaglandin F2α (PGF) treatments and the difficulty for oestrus detection in B. indicus cattle have limited the widespread application of artificial insemination (AI) and emphasizes the need for treatments that control follicular development and ovulation. Follicular-wave development in B. indicus cattle can be controlled mechanically by ultrasound-guided follicle ablation, or hormonally by treatments with GnRH or oestradiol and progestogen/progesterone in combination. Treatments with GnRH plus PGF and a second GnRH (synchronization protocol known as Ovsynch) or oestradiol benzoate (known as GPE) have resulted in acceptable pregnancy rates after fixed-time AI (FTAI) in cycling cows, but results were lower in heifers and cows in postpartum anoestrus. Alternatively, treatments with oestradiol and progestogen/progesterone releasing devices resulted in synchronous emergence of a new follicular wave, and a second oestradiol or GnRH treatment after device removal resulted in synchronous ovulation and acceptable pregnancy rates to FTAI. Furthermore, oestradiol and progesterone treatments combined with eCG (given at the time of device removal) increased pregnancy rates in suckled B. indicus cows and may be useful for the treatment of cows in postpartum anoestrus. In summary, exogenous control of luteal and follicular development facilitates the application of assisted reproductive technologies in B. indicus cattle by offering the possibility of planning AI programs without the necessity of oestrus detection and without sacrificing the overall results.

Monozygotic twinning in rhesus monkeys by manipulation of in vitro-derived embryos.

The nonhuman primate is a relevant model for human disease that can be used for diverse biomedical investigations. The ability to propagate a founder animal by application of assisted reproductive technologies is pressing, but an even greater need in many studies is access to genetically identical animals. In an effort to create genetically identical monkeys, we evaluated two approaches to monozygotic twinning; blastomere separation, and blastocyst bisection. Embryos were produced by intracytoplasmic sperm injection of oocytes recovered following controlled ovarian stimulation. The quality of demiembryos produced in these efforts was evaluated by quantitating the efficiency of creating identical pairs for embryo transfer, by morphological assessment, by the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) in the blastocyst, and by the outcome of embryo transfer to synchronized host animals. Pairs were produced in high yield (85%-95%) by both twinning methods. Demiembryos resulting from blastomere separations at the 2- or 4-cell stage grew to blastocysts at the control frequency. Demiblastocysts contained, on average, half the number of cells of the intact controls while maintaining the same ICM:TE or ICM:total cell ratio. The equivalency of demiblastocysts within a set was also evaluated by differential cell counting. Embryo transfers of identical sets led to a 33% clinical pregnancy rate, with two twin pregnancies initiated. Neither pregnancy resulted in term birth of monozygotic twins, but our results are sufficiently encouraging to justify a large-scale twinning trial in the rhesus macaque.

Reproductive endocrine patterns in captive female and male red wolves (Canis rufus) assessed by fecal and serum hormone analysis

AbstractReproductive steroid profiles in female (n=13) and male (n=5) red wolves (Canis rufus) were characterized in fecal samples collected during the breeding season (December—May) and over a 1 year period, respectively. Blood samples from females (n=12) also were collected during the periovulatory period for luteinizing hormone (LH) and steroid analysis. High performance liquid chromatography (HPLC) of fecal extracts determined that estradiol and estrone constituted the major and minor forms, respectively, of fecal estrogen metabolites. Although native progesterone was present, pregnane metabolites predominated as the major forms of fecal progestins. HPLC analysis of fecal extracts from males revealed no native testosterone, but rather the predominance of more polar androgen metabolites. Based on hormone profiles and/or pup production, females were classified as pregnant (n=3), ovulatory‐nonpregnant (n=9), or acyclic (n=3). Longitudinal monitoring of females indicated no pregnancy‐specific differences in concentrations of either fecal progestagen or estrogen metabolites compared to ovulatory‐nonpregnant individuals; however, baseline progestagen concentrations were consistently elevated in acyclic females. There was good correspondence between serum and fecal steroid concentration during the periovulatory period. A rise in serum estrogens preceded the ovulatory LH surge which was then followed by a significant progesterone rise during the luteal phase. In males, changes in fecal androgen metabolite concentrations coincided with photoperiod fluctuations, increasing in late autumn and reaching peak concentrations during mid‐ to late winter just before the start of the breeding season. Collectively, these results serve as a database of ovarian and testicular endocrine events in this species, which can be utilized in population management and application of assisted reproductive technologies. Zoo Biol 21:321–335, 2002. © 2002 Wiley‐Liss, Inc.

The control of follicular wave development for self-appointed embryo transfer programs in cattle

Our expanding knowledge of the control of follicular wave dynamics during the bovine estrous cycle has resulted in renewed enthusiasm for the prospects of precisely controlling the follicular and luteal dynamics and finely controlling the time of ovulation. Follicular wave development can be controlled mechanically by ultrasound-guided follicle ablation or hormonally by treatments with GnRH or estradiol and progestogen/progesterone in combination. Treatment of cattle with GnRH in combination with prostaglandin F 2α (PGF) 7 d later and a second GnRH 48 h after PGF (known as Ovsynch) has resulted in acceptable pregnancy rates after fixed-time AI in lactating dairy cows and in recipients in which embryos were transferred without estrus detection. Alternatively, treatments with estradiol and progestogen/progesterone-releasing devices resulted in synchronous emergence of a new follicular wave and, when a second estradiol treatment was given 24 h after device removal, synchronous ovulation and high pregnancy rates to fixe-time AI. Self-appointed embryo transfer (without estrus detection) using estradiol and progesterone treatments have resulted in pregnancy rates comparable with those obtained with recipients transferred 7 d after estrus. Furthermore, estradiol and progesterone treatments combined with PGF and eCG (given 1 d after the expected time of wave emergence) have resulted in high rates of recipients selected for transfer (84.6%) and an overall pregnancy rate of 48.7% (recipients pregnant/recipients treated). Estradiol and progestogen/progesterone treatments have also been widely used for self-appointed superstimulation protocols with equivalent embryo production to that of donor cows superstimulated using the traditional approach beginning 8 to 12 d after estrus. In summary, exogenous control of luteal and follicular development facilitates the application of assisted reproductive technologies in cattle by offering the possibility of planning the superstimulation of donors and synchronization of recipients at a self-appointed time, without the necessity of estrus detection and without sacrificing results.

Ovarian activity in the scimitar-horned oryx (Oryx dammah) determined by faecal steroid analysis.

Ultrasonography and radioimmunoassay (RIA) of serum oestradiol-17beta, luteinizing hormone (LH) and progesterone, and faecal oestrogen and progestin was used to assess ovarian activity in the scimitar-horned oryx (Oryx dammah). Ovarian examination using ultrasonography revealed maximal follicle and corpus luteum (CL) diameters of 15 and 32 mm, respectively. Steroid hormone metabolite distribution among individual faecal pellets within the same defaecation was relatively homogeneous with coefficients of variation averaging 10.2+/-1.8% and 16.2+/-4.6% for oestrogens and progestins, respectively. Elevated faecal oestrogen concentrations were associated with large (> 10 mm) antral follicles detected by ultrasonography. Periovulatory peaks in faecal oestrogen excretion, coincident with nadirs in progestin excretion, were detected in three females. Faecal progestin excretion exhibited a similar temporal pattern to serum progesterone concentrations, with a time lag of approximately 16 h. Faecal progestin concentrations corresponded with the presence of functional CL and proved useful for monitoring luteal function, spontaneous and prostaglandin-F2alpha analogue-induced luteolysis and anovulation. In summary, faecal steroid monitoring is a practical, noninvasive method for characterising ovarian steroid excretion and has potential for facilitating the application of assisted reproductive technologies in scimitar-horned oryx.

Assisted reproductive technology in nondomestic ungulates: A model approach to preserving and managing genetic diversity

The application of assisted reproductive technology (ART) to conservation biology has tremendous potential in the management and preservation of germ plasm from nondomestic ungulate species. Traditional approaches of superovulation and nonsurgical embryo recovery have been hampered in these species by inconsistent responses to commercially available gonadotropin preparations and by substantial interspecific variations in the details of reproductive regulatory processes. Although our general knowledge of reproductive physiology is improving, it appears that the production of embryos by IVF will more efficiently advance wildlife conservation efforts. This technology is already proving to be a powerful tool for rescuing gametes (sperm and oocytes) directly from the gonads of wildlife after death or gonadectomy. Other advanced procedures, like sperm microinjection and assisted hatching, may prove necessary for optimizing in vitro embryo production and in vivo developmental competence. Finally, there is a need to direct research attention to pathogen interactions with the zona pellucida (i.e. zona adherence and washing practices) to enhance the approved importation of valuable genetic material from free-ranging animals.

In vitro fertilization of pig and sheep oocytes matured in vivo and in vitro

The domestic pig is not only an economically-important livestock species, but also an increasingly recognized biomedical animal model due to its physiological similarities with humans. As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in the pig using either in vivo or in vitro production methods. The application of assisted reproductive technologies (ART) has the potential to increase reproductive efficiency in livestock. These technologies include, but are not limited to: artificial insemination (AI), fixed-time AI, embryo transfer, cryopreservation of sperm/oocytes/embryos, in vitro fertilization and somatic cell nuclear transfer (cloning). However, the application of ART is much less efficient in the pig than in many other mammalian species such as cattle. Until recently, the underlying causes of these inefficiencies have been difficult to study, but advances in molecular biology techniques for studying gene expression have resulted in the availability of a variety of options for gene expression profiling such as microarrays, and next generation sequencing technologies. Capitalizing on these technologies the effects of various ARTs on the porcine embryonic transcriptome has been determined and the impact on the related biological pathways and functions been evaluated. The implications of these results on the efficiency of ARTs in swine, as well potential consequences for the developing embryo and resulting offspring, are reviewed.