Transfer of Bovine Blastocysts Derived from Short-term In Vitro Culture of Low Quality Morulae Produced In Vivo

Introduction

Embryo quality constitutes an important factor, influencing the success of an embryo transfer program. Morphological differences between high and low quality embryos can be precisely estimated by cell ultrastructural analysis. For instance, cells of low quality morulae contain nucleoli with low transcriptional activity, a large number of cytoplasmic lipid droplets and immature mitochondria, and poorly developed junctional complexes and microvilli (Abe et al. 2002). However, ultrastructural evaluation cannot be applied to embryos destined to transfer. Thus, technicians working with embryo transfer in farm conditions, use optical microscopy to infer the quality of the embryos according to some morphological criteria, such as the darkness of the cells, the homogeneity and size of the blastomers, cytoplasmic granulation, and the degree of fragmentation of the blastomers (Overstrom 1996). Also, based on similar morphological criteria, the International Embryo Transfer Society elaborated a guide to classify embryo quality or ‘viability’ in four categories or Grades (1 = excellent or good; 2 = regular; 3 = poor and 4 = death or degenerated) (Robertson and Nelson 1998). Although a quick, simple, and not an invasive procedure, morphological evaluation is a subjective method that requires expertise in order to classify precisely embryo Grade. Even so, a study conducted by Farin et al. (1995) showed a 68.5% agreement in the embryo evaluation with several morphological defects (Grades 2 and 3). Despite the subjectivity of the morphological evaluation, there is a good relationship between the pregnancy rate and the previous classification of the embryos. Linder and Wright (1983) obtained the highest pregnancy rate with embryos of good and excellent quality. However, considering that 20% to 30% of the poor quality embryos continue their development after transfer into the recipient, it is almost impossible to determine which are the good ones without having the expenses of a transfer. Consequently, those embryos are frequently discarded, unless the recipients are exceptionally available at the day of collection.

Reichenbach (2003) suggests that a way to increase the development capacity of partially degenerated embryos is to culture them before the transfer. Recent observations established that relatively simple culture systems, such as those utilized for maintaining embryos after collection, can promote the mitotic activity and increase the total cell number in poor quality embryos after a 24-hour culture (Romo et al. 2002; Alvarez et al. 2004). However, there is no information if such reconstituted embryos may program later developments, resulting in short-term effects, particularly on gene expression and metabolism at the blastocyst stage, resulting in low pregnancy rates after the transfer into recipients.

The objective of this study was to evaluate the pregnancy rate after transfering the bovine blastocyst derived from short-term in vitro culture Grade 3 morulae produced in vivo.

Material and Methods

The study was carried out in the experimental herd of the Instituto de Zootecnia, located in Nova Odessa, São Paulo, Brazil (22°47′ Lat S and 47°18′ Lon W). Caracu and Nelore (a Bos taurus and B. indicus breed, respectively) cows were used as donors while crossbred Caracu × Nelore heifers were used as recipients. Animals were maintained in Guinea grass (Panicum maximum, Tanzania cultivar) pastures, and, during the dry season, they were supplemented with concentrate feed to attend to their nutritional requirements. Mineral salt and water were available ad libitum. All procedures for the usage of animals were approved by the Instituto de Zootecnia Animals Ethics Committee.

At an unspecific stage of the estrous cycle, cows were treated with a intravaginal device containing progesterone (CIDR-B, InterAg, New Zealand) and with an intramuscular (im) injection of 2.5 mg estradiol benzoate (Estrogin, Farmavet, Brazil). Four days later, the animals received 320 IU FSH-LH (Pluset, Calier, Spain) ministered by single subcutaneous (sc) injection in the shoulder region. In the morning of day 7, the animals received 150 mcg of D(+) cloprostenol (Veteglan, Calier, Spain) im and the CIDR-B was withdrawn in the afternoon. Donors were inseminated 48 and 62 h after the Veteglan injection. Embryo recovery was carried out by the cervical route 7 days after the insemination, and the embryos were evaluated morphologically under a dissecting microscope (60× magnification), and classified according to the criteria defined by the International Embryo Transfer Society (Robertson and Nelson 1998) by an experienced embryologist. All recovered viable (morulae and blastocyst) and unviable (oocytes and degenerated embryos) structures were taken into consideration to determine the efficiency of embryo production. However, only Grades 1 and 3 morulae were included in the experimental culture and embryo transfer data.

Individual Grade 1 morulae (n = 31) were non-surgically transferred to the uterine horn ipsilateral to the corpus luteum of recipients in the first hour after collection and served as a reference of the pregnancy rate (Group 1). Grade 3 morulae were also transferred into recipients (Group 2, n = 35), as described in Group1, or cultured in 4-well dishes (1.9 cm²/wall, Nunc, Denmark) containing 0.5 ml of Holding Plus® (Bioniche Animal Health, Belleville, Ontario, CA) (n = 58) or TCM-199 with Earle's salts, L-glutamine, and sodium bicarbonate (Nutricell, Brazil) supplemented with 10% Fetal Calf Serum (FCS, Cultilab, Brazil) (n = 44) during 24 h at 38.5°C. Morulae were incubated under high humidity with (TCM-199) or without (Holding Plus®) 5% CO₂ atmosphere. The blastocysts originated after the culture period were morphologically classified (Grades 1, 2 and 3) and transferred into recipients (Group 3, n = 47). Recipient heifers were synchronized with donors in more or less one day. Estrus synchronization of cyclic heifers was achieved injecting 150 mcg of Veteglan one day before the superovulated donors. Pregnancy was confirmed by ultra sound diagnosis 60 days after the transfer.

Results

From 38 donors submitted to the superovulation process, 31 (81.5%) having a good ovarian response (>3 CL) were collected. In total, 427 structures were recovered, from which 308 (72.1%) were viable and 119 (27.9%) were non-viable embryos. Viable embryos (morulae and blastocysts) were classified as Grade 1 (n = 78), Grade 2 (n = 79) and Grade 3 (n = 151) embryos while no viable embryos were constituted by oocytes (n = 24) and degenerate (n = 95) structures. Seventy-one per cent of the cows produced at least one Grade 3 embryo, and 29% of cows produced only Grade 3 embryos.

Medium TCM-199 was more efficient than Holding Plus® to advance the development stage to blastocyst (Table 1).

Transfer of cultured blastocyst derived from Grade 3 morulae resulted in no different pregnancy rates than control, non-cultured, Grade 1 morulae, while the pregnancy rate of non-cultured Grade 3 morulae was lower (p < 0.05) from control or cultured embryos (Table 2).

The pregnancy rate of Grade 3 cultured blastocyst was lower (p < 0.05) than cultured Grade 1 and Grade 2 blastocysts (Table 3).

Discussion

The results of the ovarian response and the embryo production of cows superovulated with a single dose of FSH by the sc route were similar to those reported by Kelly et al. (1997) and Alvarez et al. (1999). Grade 3 embryos represent approximately 49% of all recovered viable embryos. Considering that such embryos are unsuitable for either transfer or freezing, strategies to select the ones with good chances of continuing their development can be of fundamental importance for the feasibility of an embryo transfer program.

The medium TCM-199 was more efficient than Holding Plus® to support development to the blastocyst stage. This result can be expected because TCM-199 is a complex medium, elaborated to induce cell development by relatively long periods, while Holding Plus® is a medium destined to maintain the viability of embryos for a short period after embryo collection. Additionally, it is known that FCS accelerates preimplantation development of bovine embryos (Thompson et al. 1998). These results are similar to those reported previously by Romo et al. (2002) and Alvarez et al. (2004) using culture media with or without FCS. Even though the blastocyst development rate was higher with TCM-199, Holding Plus® would be an interesting medium to be used in situations in which a more sophisticated incubator maintaining a CO₂ atmosphere is not available.

The quality grade of both, fresh morulae and cultured blastocyst, was the main variable to show a significant effect on the pregnancy rate after direct transfer to the recipients. These results strongly confirm the presumption of Lindner and Wright (1983) who suggest that the morphological evaluation of bovine embryos provides accurate information for the prediction of the pregnancy rate, following embryo transfer. Those authors obtained pregnancy rates of 45%, 44%, 29% and 20% after the transfer of embryos (16 cells to hatched blastocysts) classified as excellent, good, fair and poor, respectively. Whether short-term culture can halt the development of non-viable Grade 3 morulae or enhance the quality of viable Grade 3 morulae, or both, additional studies are needed. It seems that the short-term culture of Grade 3 morulae, even under sub-optimal conditions, develops to the blastocyst stage without the perturbations in the pattern of gene expression and metabolism as observed in blastocysts derived from the bovine oocyte maturation, fertilization and long-term in vitro culture (Russell et al. 2006; Lim et al. 2007).

Despite the subjectivity, no practical method of replacing the visual morphological scoring has been developed until now. The influence of morphological deviations on the viability of apparently low and high quality embryos remains unresolved. Aguilar et al. (2002) found that approximately 50% of the embryos considered to be of good quality, under the magnifying glass of optical microscopy, presented characteristics of the degenerative stage when analysed by electron microscopy. However, differently from poor quality embryos, ultrastructural alterations in good quality embryos seem unable to compromise further embryo development. Thus, some Grade 3 morulae will halt their development during in vitro culture, while the ones that progress to the blastocyst stage have a higher chance of resulting in pregnancy when transferred into recipients. Therefore, further observation of low quality morulae during in vitro culture may confer a selection advantage during the transfer. This will obviously result in a recipient economy.

In conclusion, short-term in vitro culture of poor quality in vivo-produced morulae and the transfer of resulting blastocysts appear to increase the pregnancy rates. Such practices can contribute to the optimization of embryo transfer programs.