Elsevier

Theriogenology

Volume 95, June 2017, Pages 141-148
Theriogenology

Morphometric assessment of in vitro matured dromedary camel oocytes determines the developmental competence after parthenogenetic activation

https://doi.org/10.1016/j.theriogenology.2017.03.015Get rights and content

Highlights

  • This is the first objective morphometric classification for in vitro matured camel oocytes.

  • Classification based on extrusion of first polar body will exclude around half of the oocytes from being involved in other experiments.

  • Oocytes with no polar body but perivitelline space length <10 μm and ooplasm diameter >115 μm were able to develop in to blastocysts.

  • The classification discriminates discarded and competent oocytes to be used for parthenogenesis, ICSI and the nuclear transfer.

Abstract

The aim of the current study was to improve the selection method of camel oocytes after in vitro maturation by reducing exclusion criteria that were based only on the presence of the first polar body. A combined nuclear and morphometric assessment of camel oocytes after in vitro maturation was included to perform a judgment. The nuclear status of the oocytes, including the presence of the first polar body, meiosis I stage, and lack of nuclear materials, was investigated. The morphometric criteria that comprised the dimensions of each oocyte were as follows: diameter of the whole oocyte, including the zona pellucida (ZPO), zona pellucida thickness (ZPT), ooplasm diameter (OD), the perivitelline space (PVS) area, and PVS diameter. Among the oocytes with different nuclear status, there were no differences in ZPO and ZPT. However, oocytes with no nuclear material showed a significant reduction in OD (110.19 ± 1.4 μm) and a significant increase in PVS area (2139 ± 324.6 μm2) and PVS diameter (13.9 ± 1.96 μm) when compared with oocytes in the meiosis I stage (117.41 ± 2.85 μm, 1287.4 ± 123.4 μm2, and 8.56 ± 0.65 μm, respectively). To simplify the selection, the major difference between meiosis I and degenerated oocytes was the diameter of the PVS, which was greater than the ZPT in degenerated oocytes. Therefore, three groups were morphologically differentiated into oocytes with polar bodies (PB1), meiosis I (MI) oocytes, and degenerated oocytes. MI oocytes were able to extrude the polar body after activation but were not able to develop into blastocysts. In contrast, MI oocytes were able to develop into blastocysts after a biphasic activation protocol in which the oocytes were electrically activated and treated with ionomycin after 2 h. In conclusion, the results obtained by the morphometric assessment allowed us to develop a simple and objective classification system for in vitro matured dromedary camel oocytes, which will lead to accurate oocyte selection for the support of subsequent embryonic development.

Introduction

There are several challenges in the in vitro production of camels embryos; for instance, a limited number of slaughtered camels is available, wherein the majority of the slaughtered animals are either old, culled for infertility, or very young and have not attained maturity [1]. In Saudi Arabia, the collection of ovaries from fertile camels is particularly challenging, because there are restrictions on fertile she-camel slaughtering, as given in the regulations of Ministry of Municipal and Rural Affairs in Kingdom of Saudi Arabia [2]. Additionally, the physiological complexity of follicle maturation during the follicular waves might be associated with the asynchronous maturation of oocytes retrieved from different follicular stages in the camel [3], [4], [5].

Therefore, the careful morphological selection of camel oocytes is a crucial step in the prediction of the subsequent developmental competence and to avoid the disposal of indispensable oocytes, especially when the available oocytes are limited. The morphometric selection of in vitro matured oocytes has been reported in humans and several animal species [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. To our knowledge, no studies have previously utilized the morphometric classification for in vitro matured oocytes in camels.

Remarkably, a prerequisite to enable the developmental competence of the oocyte is the successful completion of meiosis. One main criterion in the judgment of oocyte maturation is the completion of meiosis I and the extrusion of the first polar body after asymmetric cell division with the vast majority of the cytoplasmic volume conserved for the oocyte [17]. After completion of the first meiotic division, the oocyte enters meiosis II and remains arrested in metaphase II [18]. Specifically, several reports revealed that approximately 50% of camel oocytes can extrude the first polar body after an in vitro maturation period [19], [20], [21], [22], [23]. Hence, approximately half of the cultured oocytes will be excluded from subsequent experiments, such as parthenogenesis, intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, which use denuded oocytes. Additionally, the ooplasm diameter was associated with the developmental competency of the oocytes and considered as an essential factor for oocyte selection in humans and a variety of animal species [9], [24], [25].

Accordingly, the current study was carried out to determine several morphometric dimensions of camel oocytes, such as the diameter of the ooplasm and the perivitelline space in relation to the nuclear status after in vitro maturation. In addition, the study aimed to examine the effect of assisted activation on the oocytes that were morphometrically acceptable but did not extrude the polar body, to increase the yield of oocytes that could support early embryonic development.

Section snippets

Chemicals

Unless otherwise stated, all chemicals and hormones were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA).

Collection of ovaries, cumulus-oocyte complexes (COCs), and in vitro maturation (IVM)

Camel ovaries were obtained from a local abattoir in Riyadh and transported in 0.9% (v/v) NaCl solution at 30–33 °C to the laboratory within 4–6 h. The follicular contents from antral follicles (2–8 mm in diameter) were aspirated using an 18-ga needle attached to a 10 mL-disposable syringe. Cumulus-oocyte complexes (COCs) with evenly granulated cytoplasm that were enclosed by more

Nuclear status of the in vitro matured oocytes

The total in vitro matured oocytes (n = 135, 5 replicates) showed three patterns of nuclear status (Fig. 2): oocytes with the first polar body extruded (52.3 ± 2.1%), oocytes without polar body but with the nucleus in different stages of meiosis I (i.e. metaphase I and anaphase I stages; Fig. 2) (22.6 ± 3.4%), and oocytes with no nuclear materials (19.1 ± 1.4%). Oocytes with abnormal morphology or with clear damage to the ooplasm or oolemma architecture were excluded from the experiments

Discussion

In this comprehensive study, we provided an objective morphometric assessment of camel in vitro matured oocytes in order to select and utilize more oocytes that have the capability to support embryonic development.

Recent studies reported the inefficiency of camel oocyte in vitro maturation with only 25%–40% of the oocytes extruding the first polar body [22], [23]. However, IVM results showed that 52% of the oocytes extruded the first polar body, which was in agreement with previous studies [1],

Conclusion

In conclusion, the current study provided an objective morphometric assessment of camel in vitro matured oocytes and this morphometric classification discriminated between the discarded and competent oocytes. This result would increase the available competent oocytes that could be used for experiments dealing with denuded mature oocytes such as parthenogenesis, ICSI, and nuclear transfer.

Disclosure statement

All authors declare no conflicts of interest regarding the publication of this paper.

Acknowledgements

The authors extended their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the research group No (RG-1438-018).

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