Embryo development of prepubertal goat oocytes fertilised by intracytoplasmic sperm injection (ICSI) according to oocyte diameter
Introduction
The success of in vitro fertilisation (IVF) is dependent on both sperm maturation and capacitation for penetration of the oocyte and on oocyte quality to support embryo development. Unfortunately, IVF does not always provide good results because of male infertility or high fertilisation abnormalities (such as polyspermy) found using this procedure. In order to bypass this step and minimize variability due to sperm capacitation and penetration, intracytoplasmic sperm injection (ICSI) could be used as a technique for sperm and oocyte quality evaluation studies.
The ICSI is a fertilisation technique that involves mechanical injection of a single sperm into the oocyte. This technique was first reported in sea urchins [1] but only when Uehara and Yanahimashi [2] obtained good results in hamsters, was ICSI tested with success in other species (mice [3]; sheep [4]; horse [5]; pig [6]; cattle [7]; goats [8], [9]). The success of ICSI depends on the protocol used for each species. In some species, fertilisation and developmental rates obtained with ICSI have been low, probably due to inappropriate oocyte activation or sperm capacitation that results in non-pronuclei formation. Many studies have been done on these subjects: in mice [3], rabbits [10], sheep [11] and horses [12]; ICSI by itself are sufficient for oocyte activation, sperm head decondensation and embryo development. In contrast, in cattle and pigs, ICSI was combined with chemical activation (ethanol [13]; Ca-ionophore [7], [14]; ionomycin alone [15], [16]; or combined with 6-dimethilaminopurine (6-DMAP [15], [16], [17]) or electrical activation [14], [18], [19] in order to improve activation of oocytes. Piezo-drill ICSI was reported to increase both activation and cleavage rates in horses [12], [20], goats [9] and cows [21], [22], [23].
It is known that a permeabilisation treatment of the sperm membrane prior to ICSI helps the decondensation of head sperm. Equine [24] and cattle [25] have very stable sperm membranes that require cryopreservation or a strong promoter of spermatozoa capacitation such as ionomycin, which destabilizes plasma membranes thus increasing fertilisation rates. In goats, a stronger sperm treatment using heparin plus ionomycin improved in vitro fertilisation and embryo development results, both in adult [26] and prepubertal goats [27]. In our previous study, using fresh semen and manual needle injection ICSI, prepubertal goat oocytes needed chemical activation (ionomycin plus 6-DMAP) to be fertilised. However, chemical treatment also increased parthenogenetic embryos [28]. In order to avoid oocyte chemical activation, in the present study we will use a sperm pretreatment using heparin plus ionomycin before ICSI to destabilise the membrane so as to help sperm head decondensation.
The ICSI technique improved embryo development of prepubertal goat oocytes compared to the IVF protocol [28]. However, embryo development was low in both cases. Several studies have shown reduced embryo development of oocytes from prepubertal females (reviewed by [29]). In adult females, a positive relationship has been found between follicle diameter, oocyte diameter and the developmental competence of oocytes (reviewed by [30]). In cattle, follicles larger than 6 mm [31] provided the most competent oocytes. Moreover, other studies obtained the most competent oocytes when their diameter was 135 μm [32], [33]. Cattle oocyte diameter seems to be smaller than that of goat oocytes. Thus, bovine full meiotic competence is achieved with a follicle size of about 3 mm, which corresponds to an oocyte diameter of about 110 μm [32]. In cattle, the relationship between cleavage rate and oocyte diameter has been evaluated [34]: cleavage rates of 7, 41 and 55% were obtained for bovine oocytes smaller than 100, 100–109 and 100–119 μm, respectively, and this percentage increased to 71% when oocytes were bigger than 120 μm. When embryo development was studied, oocytes smaller than 100 μm and from 100 to 109 μm triggered the lowest blastocyst rates (20 and 30%, respectively) while oocytes from 100 to 129 μm triggered the highest percentage (60%). There was a small decrease in blastocyst percentages when oocytes measured more than120 μm (49%).
In cattle [35] and sheep [36], oocytes from prepubertal females were found to be smaller than oocytes from adult females. In goats, de Smedt et al. [37] showed that adult goat oocytes sequentially acquired meiotic competence in follicles ranging from 0.5 to 2–3 mm in diameter, and Crozet et al. [38] observed that the mean oocyte diameter increased from 96 ± 0.3 to 136 ± 0.6 μm as follicle size increased from 0.5 to 2–3 mm. In prepubertal goat oocytes, Martino et al. [39] also observed that meiotic competence acquisition was achieved with a follicle diameter of 2–3 mm with an oocyte diameter of 134 ± 5.37 μm. In both, adult [37] and prepubertal goat oocytes [39], these authors observed that oocytes smaller than 110 μm corresponded to incompetent meiotic oocytes, from 110 to 125 μm they corresponded to partially competent oocytes and oocytes larger than 125 mm had full meiotic competence. No studies about the relationship between prepubertal goat oocyte diameter and embryo development have been carried out.
Thus, the aim of the present study was to determine embryo development competence of prepubertal goat oocytes according to their diameter and fertilisation by ICSI after a spermatozoa treatment with ionomycin and heparin.
Section snippets
Recovery and in vitro maturation of oocytes
Ovaries from prepubertal goats (1–2 months old) were obtained from a local abattoir and transported to the laboratory in PBS solution Dulbecco's phosphate-buffered saline (PBS, P-4417, Sigma Chemical Co., St. Louis, MO, USA) with 50 (g/ml of gentamycin sulphate at 38.5 °C. The ovaries were rinsed with the same solution. The cumulus-oocyte complexes (COCs) were recovered after slicing the ovaries submerged in slicing medium: TCM199 (Sigma, M-2520), supplemented with 2.2 mg/ml NaHCO3, 2% (v/v) steer
Experiment 1
Table 1 shows the nuclear stage of oocytes at 17 h after IVF, ICSI and Sham injection.
The ICSI group provided 40.9% of normal fertilised zygotes (2 PN), thus percentage decrease to 26.6% in IVF group (P < 0.05). Abnormalities in fertilisation were observed both in ICSI and IVF groups without statistically significant differences. The percentage of 3 PN was 2.6 and 5.3% for ICSI and IVF, respectively, and the percentage of 1 PN was 6.9 and 0.3%, respectively. When we evaluate the origin of the
Discussion
The present study shows that conventional ICSI with a previous treatment of sperm cells combining ionomycin and heparin as capacitators, increases embryo development and blastocyst rates in prepubertal goat oocytes compared to previous studies where spermatozoa were only capacitated with heparin [28]. In agreement with other studies in cattle [16], [23], [45], pretreatment of sperm cells could be useful in the development of ICSI embryos. In the present study, we also show that the percentage
Acknowledgment
Grant sponsor MEC; Grant number: AGL2004-04737-CO3-01-GAN.
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