Ovum Pick-Up (OPU) in Cattle: An Update

SALVADOR RUIZ LOPEZ

Department of Physiology, Faculty of Veterinary Science,

International Excellence Campus for Higher Education and Research "Campus Mare Nostrum", University of Murcia, Murcia, Spain

Institute for Biomedical Research of Murcia (IMIB-Arrixaca),

Murcia, Spain

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ABSTRACT

The technique of ultrasound-guided transvaginal follicular aspiration for ovum pick-up (OPU) is a non-invasive procedure for recovering oocytes from antral follicles in live animals, especially in cows and mares. It was originally developed for assisted reproduction in the human species to assist infertility and was used for the first time in cattle in the Netherlands at the end of the decade of the 1980s. OPU does not interfere with the normal reproduction and production cycles of the donor and can be used in adult cows in various physiological states, in old annuals with reproductive disorders of non-genetic origin and calves and heifers from 6 months of age. The repeated recoveiy of oocytes through OPU allows us to obtain the highest possible offspring of animals with high genetic value and speed up processes of animal selection and genetic improvement, while it is an extraordinary source of oocytes for cloning and transgenesis. It has been shown to be a feasible and practical alternative to the МОЕТ (Multiple Ovulation and Embryo Transfer) program and it is being more and more used for commercial applications in the world. However, we must bear in mind that the implementation of an OPU program always requires the support of a specialized laboratory for embryo production.

The aim of this chapter is to describe the most important aspects of the OPU procedure used in cattle considering its historical development, the actual situation of OPU worldwide, the OPU equipment and procedure, technical and biological factors influencing OPU results, other uses of OPU, potential risks and most common sequelae, a brief review about OPU in buffaloes, and finally some considerations about the future of OPU/IVEP (in vitro embryo production).

INTRODUCTION

The association of reproductive efficiency and genetic selection is strategic for the success of daily and beef industries. Reproductive technologies, such as ovum pick-up (OPU) and in vitro embryo production (IVEP), can rapidly enhance genetics of cattle through both female and male linaje (Watabane et al., 2017).

Puncture and aspiration of bovine ovarian follicles have been used for several decades to retrieve oocytes for IVEP. Cumulus oocyte complexes (COCs) can be recovered from the ovaries of both slaughtered cows or living donors. The method of oocyte retrieval has an impact on COC morphology, and the importance of “good” quality oocytes as the primaiy pre-requisite for success in oocyte maturation and in vitro development has been considered (Merton et al., 2003). From a practical reproductive perspective, aspiration of immature follicles is particularly interesting when performed on living donors, because the procedure can be repeated, and is highly repeatable (Bols and Stout, 2018). Effectively, one of the fundamental conditions for successful commercial IVEP is the development of an efficient system that allows the recovery of oocytes of living donors of known genetic value that can be used several times with minimal consequences for the animals (Da Silva et al., 2016). The repeated recoveiy of oocytes through OPU allows to obtain the highest possible offspring of annuals with high genetic value and speed up processes of animal selection and genetic improvement (paternal and maternal way), while it is an extraordinary source of oocytes for cloning and transgenesis (Ding et al., 2008).

The technique of ultrasound-guided transvaginal follicular aspiration for OPU is a non-invasive procedure for recovering oocytes from antral follicles in live animals, especially in cows and mares. It was originally developed for assisted reproduction in the human species to assist infertility (Lenz and Lauritsen, 1982), and was used for the first time in cattle in the

Netherlands at the end of the decade of the 80s (Pieterse et al., 1988). The use of OPU routinely in veterinary assisted reproduction began in 1994 (Kruip et al., 1994).

Unlike conventional Multiple Ovulation and Embryo Transfer (МОЕТ), OPU does not interfere with the normal reproduction and production cycles of the donor. It is a technique that can be used in adult cows in various physiological states (cyclical, non-cyclic, in the first third of gestation and in which they do not respond to honnonal stimuli), in old animals with reproductive disorders of non-genetic origin (Galli et al., 2001), in calves and heifers from 6 months (mo) of age (Taneja et al., 2000), and even soon, annuals after calving (2-3 weeks) could be a suitable donor. It has been shown to be a feasible and practical alternative to the МОЕТ program (Bousquet et al., 1999), and it is being more and more used for commercial applications in the world (Pontes et al., 2011). However, we must bear in mind that the implementation of an OPU program always requires the support of a specialized laboratory for an embryo production. Moreover, because of the economic value of the calves bom, adequate veterinary assistance is recommended to minimize losses owing to the possible incidence of the large offspring syndrome (LOS) or other common perinatal pathologies (Galli et al., 2014).

Altogether, the cost of producing an embryo by OPU in daily cows in Europe could be 50-100% greater than by МОЕТ. This greater cost and the current breeding context in Europe allow for the use of OPU for a veiy specialized niche market. Different conditions in other countries offer different opportunities also dictated by economics: for example, the large use in Brazil is certainly determined by the fact that OPU in general works better and it is more cost effective than superovulation in Bos indicus beef donors (Galli et al., 2014).

The aim of this chapter is to describe the most important aspects of the OPU procedure used in cattle considering its historical development, actual situation of OPU worldwide, the OPU equipment and procedure, technical and biological factors influencing OPU results, other uses of OPU, potential risks and most common sequelae, a brief review about OPU in buffaloes, and finally some considerations about the future of OPU/IVEP.

HISTORICAL DEVELOPMENT

The first developed in vivo oocyte retrieval procedure was laparotomy that provides full access to the abdominal cavity for the direct manipulation of the internal organs. Despite easy access to the ovaries, all surgery risks were present, like anesthesia, contamination, and problems performing the same procedure several times. Other possibilities were also considered in the past, including colpotomy, small incision in the vagina fornix, with usual risks of surgical procedure and clear limitations, and laparoscopy, via either the transvaginal or paralumbar approach, comparatively less invasive method; however, despite its promising prospects, many articles described teclmical difficulties in addition to the occurrence of fibroids and ovarian adhesions (reviewed by Da Silva et al., 2016).

All of the oocyte recovery-related challenges in cattle were considered in a new prospective analysis of its successful in women. Effectively, most of the new methodologies proposed in live annuals represent adaptations to novelties, which have been developed in human. Of course, these techniques have been adapted in relation to their use and application in animals.

Initially, mature human oocytes were recovered ultrasonically guided percutaneous aspiration through the abdomen under local anesthesia in early 1908s (Lenz and Kauritsen. 1982); subsequently, ultrasound guided transvaginal oocyte aspiration was developed in humans (Dellenbach et al., 1984).

Repeated in vivo oocyte collection in cattle was first performed by Canadian researchers who used endoscopy via the right paralumbar fossa (Lambert et al., 1983). Callesen et al. (1987) were the first to use ultrasonography to collect oocytes from living cattle, using an ultrasonographic transducer equipped with a needle guide via a transcutaneous approach. This study was carried out in seven superovulated heifers. By rectal palpation, ovaries and follicles were visualized by ultrasound examination. A total number of 38 follicles were transcutaneously punctured and 16 oocytes were collected that resulted in a recovery rate of 42% (RR = number of oocytes collected/100 follicles punctured) and

2.3 oocytes/heifer.

In vivo oocyte collection by OPU was first established in cattle by a Dutch team, modifying a transvaginal ovum pick-up technique originally developed for use in human reproduction and in cattle. Pieterse et al. (1988) added an extension device to a convex ultrasound transducer that allowed it to be manipulated outside the bovine vagina and enabled nonsurgical oocyte recovery in cattle for the first tune. A big advantage of the transvaginal approach in cattle is that it is possible to both secure and manipulate the ovary per rectum so that it can be moved around the ultrasound transducer and needle, to present the most optimal position for puncture. In this study, OPU was performed once a week in 10 cows for a total number of 36 transvaginal aspiration procedures, during which 54 oocytes were recovered from 197 punctured follicles. The mean RR was 27.4% and the number of oocytes/cow/sampling was 1.5. The stimulation of the ovaries with pregnant mare serum gonadotropin (PMSG) increased both RR (40 vs. 18%) and the number of oocytes/cow/sampling (2.7 vs. 1.0). The estrous cycle of these animals was not interrupted due to OPU procedure on the basis of plasma progesterone measurements (reviewed by Boni, 2012). As a result, a minimally invasive method with high repeatability (Pieterse et al., 1991) for repeated oocyte retrieval from living donor cows became available.

Another possibility to collect oocytes from living cows was proposed by Reichenbach et al. (1994), who developed a laparoscopic procedure of oocyte collection (L-OPU). This technique allows the repeated laparoscopic examination of the internal reproductive organs of cows and heifers through the vaginal fornix and visually assisted follicle aspiration. L-OPU showed several advantages with respect to OPU; in particular, the aspiration of primarily superficial follicles; the direct view of the ovary; and the aspiration procedure and a reduced risk of injury to the ovary. L-OPU and OPU techniques were compared by different researchers. Becker et al. (1996) compared transvaginal OPU under ultrasonographic guidance with oocyte retrieval by endoscopic instruments. They concluded that the use of ultrasound resulted in better quality COCs, although it is not entirely clear why endoscopic aspiration should cause more damage to the COCs. Santl et al. (1998) made a comparison of ultrasound-guided (U-OPU) vs. L-OPU in Simmental heifers. These researchers found higher proportion of class I oocytes (Grade 1 or A; Oropeza et al., 2004) after U-OPU more than that after L-OPU; these differences also were reflected by the cleavage rate, and morulae and blastocysts rates, attributing to the greater changes in vacuum pressure during L-OPU vs. U-OPU as responsible for the difference in oocyte quality.

Finally, as a consequence OPU procedure was easy, repeatable, featured minimal risks, and was developed as a successful technique for retrieving oocytes from selected heifers and cows of high genetic merit (Kruip et al., 1994), to breed large numbers of calves with known production traits, and to shorten the generation interval in cattle breeding programs. Moreover, OPU allowed to obtain embryos in situations of extraovarian infertility that result in the birth of healthy animals, confirming that most cows classified as infertile are able to produce viable gametes (Seneda et al., 2001).

Indeed, the ultimate aim was to produce more embryos and pregnancies per donor cow than was possible through МОЕТ programs (Pieterse et al., 1991). Some advantages of OPU/IVEP over МОЕТ are that multiple offspring for nearly eveiy donor cow can be produced within a limited period, the number of offspring per unit time is significantly larger, and the technology is less dependent on the reproductive status of the donor cow. In addition, several bulls can be used for in vitro fertilization (IVF) on oocytes from one collection, rather than one bull as in МОЕТ, thus maximizing genetic gain and minimizing inbreeding (van Wagtendonk-de Leeuw, 2005).

OPU/IVEP, however, requires a more sophisticated and expensive laboratory setting compared to МОЕТ, and the costs per OPU/IVEP embryo are approximately twice those of МОЕТ embryos. Although OPU/IVEP has clear advantages over МОЕТ and artificial insemination (AI) in terms of number of offspring that can be produced per cow per time span (on average for AI 1 calf/year, for МОЕТ 20-25 calves/year, and for OPU/IVEP 80-100 calves/year). The efficiency of reproductive technologies in terms of number of offspring per 100 immature oocytes declines significantly with increased levels of artifice of the technologies, from AI (with 55 live calves on the ground 1 week after birth), МОЕТ (28 calves), until OPU/IVEP (with 11 calves). At the same time, problems associated with calves in terms of birth weight, congenital abnormalities, and perinatal mortality increase. This relative low efficiency in the production of healthy calves together with the relative high cost of compared to МОЕТ in cattle makes its use justifiable mainly for breeding companies and breeders, where the benefits in terms of semen sales from resulting high genetic bulls may outweigh the costs (reviewed by van Wagtendonk-de Leeuw, 2005).

Pontes et al. (2009) investigated why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, and compared embryo yield and pregnancy rate between these methods in the same Nelore (B. indicits) donor cows. The average number of embiyos produced by OPU/IVEP (9.4 ± 5.3) was higher than the МОЕТ method (6.7 ± 3.7). However, pregnancy rates were lower following transfer of in vitro produced (IVP) (33.5%) vs. in vivo derived (IVD) embiyos (41.5%). They concluded that in Nelore cows, with an interval of 15 days between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional МОЕТ.

 
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