Review: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods.

In vitro production (IVP) of embryos and associated technologies in cattle have shown significant progress in recent years, in part driven by a better understanding of the full potential of these tools by end users. The combination of IVP with sexed semen (SS) and genomic selection (GS) is being successfully and widely used in North America, South America and Europe. The main advantages offered by these technologies include a higher number of embryos and pregnancies per unit of time, and a wider range of potential female donors from which to retrieve oocytes (including open cyclic females and ones up to 3 months pregnant), including high index genomic calves, a reduced number of sperm required to produce embryos and increased chances of obtaining the desired sex of offspring. However, there are still unresolved aspects of IVP of embryos that limit a wider implementation of the technology, including potentially reduced fertility from the use of SS, reduced oocyte quality after in vitro oocyte maturation and lower embryo cryotolerance, resulting in reduced pregnancy rates compared to in vivo-produced embryos. Nevertheless, promising research results have been reported, and work is in progress to address current deficiencies. The combination of GS, IVP and SS has proven successful in the commercial field in several countries assisting practitioners and cattle producers to improve reproductive performance, efficiency and genetic gain.

Effect of different mini-volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer-assisted semen analyzer.

Straws of sex-sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106  sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex-sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane-coated silica colloid dilutions and layering configurations during centrifugation of sex-sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex-sorted sperm samples were centrifuged using mini-volume single-layer (40%, 60% and 80%) and mini-volume two-layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm® . A single layer of 40% PureSperm® recovered significantly more sex-sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two-layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single-layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini-volume single layer of 80% PureSperm® was determined to be an effective alternative to a two-layer centrifugation configuration for sex-sorted sperm selection. In addition, single-layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.

Hormonal treatments for increasing the oocyte and embryo production in an OPU-IVP system.

The objective was to enhance the inherent developmental ability of bovine oocytes retrieved by ultrasound-guided transvaginal aspiration. Various hormonal regimes were utilized to produce partially matured oocytes in vivo, in order to improve embryo development following IVF. In the first experiment, a two-by-two factorial design was used with FSH (multiple versus single dose) and im administration of LH (yes versus no) 6h prior to OPU. In all protocols (which lasted for nine consecutive weeks), ovarian stimulation was performed in the presence of a CIDR. One FSH administration was adequate for ovarian stimulation (9.33+/-0.7 and 10.14+/-0.7 follicles per cow per OPU session); however, multiple injections increased (P<0.05) follicular response (12.97+/-0.7 and 13.97+/-0.7). In the second experiment, a two-by-two factorial design was used to compare the effects, during ovarian stimulation, of the presence or absence of CIDR, and iv treatment with LH 6h prior to OPU (yes versus no), on oocyte competence (judged by blastocyst development rates following IVF). Presence of CIDR during superstimulation had no effect on the follicular response. Administration of LH 6h prior to OPU increased (P<0.05) the oocytes of higher morphological grades, and in the absence of a CIDR, improved (P<0.05) blastocyst development rate. Treatment with LH, 6h prior to OPU without the use of CIDR during ovarian stimulation, resulted in 2.89+/-0.4 blastocysts per cow per OPU session as compared to 1.56+/-0.4, 1.56+/-0.4 and 1.33+/-0.4 for all other groups. In conclusion, compared to single administration, multiple FSH administration increased (P<0.05) available follicles for aspiration. Moreover, when ovarian stimulation in the absence of CIDR was followed by administration of LH 6h prior to OPU, it increased (P<0.05) the number of blastocysts per OPU session.

Comparison of different transvaginal ovum pick-up protocols to optimise oocyte retrieval and embryo production over a 10-week period in cows.

The objective was to develop a simple and effective ovum pick-up (OPU) protocol for cows, optimised for oocyte harvest and subsequent in vitro embryo production (IVP). Five protocols differing in collection frequency, dominant follicle removal (DFR) and FSH stimulation were tested on groups of three cows each, over an interval of 10 consecutive weeks. Performance was evaluated on per OPU session, per week and pooled (3 cowsx10weeks) basis. Among the non-stimulated groups, on a per cow per session basis, once- or twice-weekly OPU had no effect on the mean (+/- S.E.M.) number of follicles aspirated, oocytes retrieved and blastocysts produced (0.6+/-0.8 and 0.7 +/- 0.7, respectively). However, DFR 72 h prior to OPU almost doubled blastocyst production (1.2 +/- 1.3). In stimulated groups, FSH treatment (80 mg IM and 120 mg SC) was given once weekly prior to OPU. Treatment with FSH, followed by twice-weekly OPU, failed to show any synergistic effect of FSH and increased aspiration frequency. When FSH was given 36 h after DFR, followed by OPU 48 h later, more (P < 0.05) follicles (16.0 +/- 5.0), oocytes (10.6 +/- 4.5) and embryos (2.1 +/- 1.2) were obtained during each session, but not on a weekly basis. Pooled results over 10 weeks showed an overall improved performance for the treatment groups with twice-weekly OPU sessions, due to double the number of OPU sessions performed. However, the protocol that consisted of DFR, FSH treatment and a subsequent single OPU per week, was the most productive and cost-effective, with potential commercial appeal.

Agro-economic impact of cattle cloning.

The purpose of this paper is to review the economic and social implications of cloned cattle, their products, and their offspring as related to production agriculture. Cloning technology in cattle has several applications outside of traditional production agriculture. These applications can include bio-medical applications, such as the production of pharmaceuticals in the blood or milk of transgenic cattle. Cloning may also be useful in the production of research models. These models may or may not include genetic modifications. Uses in agriculture include many applications of the technology. These include making genetic copies of elite seed stock and prize winning show cattle. Other purposes may range from "insurance" to making copies of cattle that have sentimental value, similar to cloning of pets. Increased selection opportunities available with cloning may provide for improvement in genetic gain. The ultimate goal of cloning has often been envisioned as a system for producing quantity and uniformity of the perfect dairy cow. However, only if heritability were 100%, would clone mates have complete uniformity. Changes in the environment may have significant impact on the productivity and longevity of the resulting clones. Changes in consumer preferences and economic input costs may all change the definition of the perfect cow. The cost of producing such animals via cloning must be economically feasible to meet the intended applications. Present inefficiencies limit cloning opportunities to highly valued animals. Improvements are necessary to move the applications toward commercial application. Cloning has additional obstacles to conquer. Social and regulatory acceptance of cloning is paramount to its utilization in production agriculture. Regulatory acceptance will need to address the animal, its products, and its offspring. In summary, cloning is another tool in the animal biotechnology toolbox, which includes artificial insemination, sexing of semen, embryo sexing and in vitro fertilization. While it will not replace any of the above mentioned, its degree of utilization will depend on both improvement in efficiency as well as social and regulatory acceptance.

Commercialization of animal biotechnology.

Commercialization of animal biotechnology is a wide-ranging topic for discussion. In this paper, we will attempt to review embryo transfer (ET) and related technologies that relate to food-producing mammals. A brief review of the history of advances in biotechnology will provide a glimpse to present and future applications. Commercialization of animal biotechnology is presently taking two pathways. The first application involves the use of animals for biomedical purposes. Very few companies have developed all of the core competencies and intellectual properties to complete the bridge from lab bench to product. The second pathway of application is for the production of animals used for food. Artificial insemination (AI), embryo transfer, in vitro fertilization (IVF), cloning, transgenics, and genomics all are components of the toolbox for present and future applications. Individually, these are powerful tools capable of providing significant improvements in productivity. Combinations of these technologies coupled with information systems and data analysis, will provide even more significant change in the next decade. Any strategies for the commercial application of animal biotechnology must include a careful review of regulatory and social concerns. Careful review of industry infrastructure is also important. Our colleagues in plant biotechnology have helped highlight some of these pitfalls and provide us with a retrospective review. In summary, today we have core competencies that provide a wealth of opportunities for the members of this society, commercial companies, producers, and the general population. Successful commercialization will benefit all of the above stakeholders.