Análisis descriptivo de las variables

Although live births derived from embryos produced through ICSI have been reported in cattle (Goto et al., 1990; Hamano et al., 1999), the efficiency of ICSI in cattle is far inferior compared to humans (Van Steirteghem et al., 1993a) and mice (Kimura and Yanagimachi, 1995). This is because bovine sperm nucleus is structurally stable due to its richness in protamine disulfide bonds, and this stability is a drag on sperm head decondensation and male pronuclear formation (Rho et al., 1998a).

Oocyte activation is the restoration of metabolic activity in the quiescent oocyte or the process of releasing the oocyte from the second meiotic arrest when the sperm fertilises it (Yanagimachi, 1994). The very early cellular event observed in all activated mammalian oocytes is an intracellular rise in Ca²⁺ concentration. The site of Ca²⁺ release and sequestration is thought to be the endoplasmic reticulum, where inisitol 1,4,5-triphosphate (IP3) reseptors are present (Kline and Kline, 1992). The first Ca²⁺ transient is followed by a series of shorter Ca²⁺ transients of high amplitude.

As fertilisation progresses, the amplitude and frequency of the Ca²⁺ transients decrease, while duration increases until and absolute cessation of Ca²⁺ oscillations

during entry into interphase and PN formation, several hours after sperm entry (Jones at al., 1995).

Unlike in human and mouse ICSI, the operation of sperm injection and the presence of a sperm in the ooplasm can not provide enough stimulation to provoke oocyte activation in cattle (Rho et al., 1998b). To solve the problems, attempts have been made to induce sperm decondensation before ICSI and to activate oocyte after ICSI. Compared to human and mouse sperm, bovine ooplasm is opaque and the bull sperm head is large. This leads to extreme difficulty in proper delivery of a sperm into an ooplasm and an increase in the volume of vehicle solution injected into oocytes. As a result, a delivered sperm might not be in the ooplasm, or if it is there, the sperm might be surrounded by enough vehicle solution to prevent it from incorporating with the ooplasm. In that case, no good result can be expected (Wei and Fukui, 2000).

Technical improvement by clarifying the oocyte cytoplasm by polarise the ooplasmic lipid and provide fine visibility for the ICSI operation by centrifugation at 6,000x g for 7 minutes (Wei and Fukui, 2000), 5 minutes Rho et al., 1998a; Chung et al., 1999). This technique also helpful in minimising the volume of PVP solution injected into oocytes. Cutting the sperm tail and reducing the PVP concentration can significantly benefit in ICSI cattle (Wei and Fukui, 2000). Table 2.2 shows ICSI in cattle.

There are several reports on the birth of healthy offspring in the human and mice after the transfer of embryos produced by ICSI without any additional oocyte activation treatment (Teserik and Mendoza, 2003; Yanagimachi, 1998). However, in cattle, there are a few reports on the birth of calves following ICSI (Goto et al., 1990;

Hamano et al., 1999; Horiuchi et al., 2002; Wei and Fukui, 2002; Galli et al., 2003b).

Table 2.2: ICSI in cattle

Author Result

Wasthusin et al., 1984 Cleavage (4%) Kameyama et al, 1985 Fertilisation (13%) Keefer et al. 1990 Cleavage (38%)

Goto et al. 1990 Cleavage (12%), offspring Heuwieser et al. 1992 Fertilisation (39%)

Pavasuthipaisit et al. 1994 Cleavage (67%), blastocyst (9%)

Rho et al. 1998a Cleavage (61%), blastocyst (24%), pregnancy (38%) Horiuchi et al. 1999 Cleavage (72%), blastocyst (28%)

Chung et al., 1999 Cleavage (62.1%), blastocyst (3.0%) Katayose et al., 1999 PN (85%), cleavage (72%)

Wei and Fukui, 2002 PN (86.3%), cleavage (71.8%), blastocyst (22.7%), ET (live born)

Horiuchi et al., 2002 Cleavage (72%), blastocyst (20%), offspring (50%) Keskintepe et al., 2002 PN (74%), cleavage (63.3%), blastocyst (29.6%) Galli et al., 2003b PN (88.6%), cleavage (59.7%), blastocyst (6.1%)

PN (88.9%), cleavage (58.5), blastocyst (7.3%), offspring (9.1%)

Li et al., 2004 Cleavage (83%), blastocyst (25%),

Rho et al., 2004 PN (54.8), cleavage (70%), blastocyst (16.3%) Fujinami et al., 2004 Cleavage (51%), blastocyst (14%)

Oikawa et al., 2005 Cleavage (75.6%), blastocyst (39.4%), offspring (47.4%)

Kato and Nagao, 2009 Cleavage (68.7%), blastocyst (20.9%)

In these species, fertilisation and cleavage after sperm injection is very low and in vitro development is very limited.

Oocyte activation is characterised by a dramatic rise in intracellular calcium concentration, which in mammals take the form of calcium oscillations (Stricker, 1999; Hafez et al., 2004) driven by an elevation in inositol triphosphate concentrations (Rice et al., 2000). The causative agent of these oscillations is proposed to be a recently described phosphoinositide-specific phospholipase C, PLC-X, which is a soluble sperm factor delivered to the oocyte following membrane fusion (Saunders et al., 2007).

Mechanical process of injection can provide an activating stimulus in some species, less than 5% of sham-injected bovine oocytes have been activated (Keefer et al., 1990; Rho et al., 1998a,b). Although in vitro matured bovine oocytes can be activated by sperm following in vitro fertilisation, resulting in production of developmentally competent embryos, these unaged oocytes have proven difficult to activate parthenogenetically (Susko-Parrish et al., 1994). When bovine oocytes are exposed to a single chemical (e.g., calcium ionophore) or electrical stimulus, which induce a transient rise in intracellular calcium, the levels of histone kinase briefly decrease but subsequently return to pre-stimulus concentrations (Collas et al., 1993).

In fertilised oocytes, however, multiple calcium oscillations are observed, and histone kinase levels remain low until the subsequent cell cycle commences (Liu and Yang, 1999; Sun et al., 1994). Treatment of oocytes with multiple calcium stimuli or a single stimulus followed by DMAP, an inhibitor of phosphorylation, or cycloheximide, an inhibitor of protein synthesis, can prevent the rise in histone

kinase and permit pronuclear formation (Susko-Parrish et al., 1994; Soloy et al., 1997).

Studies with ethanol (Nagai, 1987) or ionornycin (Ware et al., 1989) indicate that the activation rates are dependent upon the age of oocytes. Nagai (1987) reported that bovine oocyte activation with ethanol approaches peak efficiency around 27 h of maturation in vitro. The time course for responsiveness to ethanol activation is similar to the time course reported by Ware et al. (1989) for both ionomycin and electrical pulse. However, although activation rates are higher in aged oocytes than in young oocytes, it is generally accepted that aged oocytes are beyond their normal fertilisable life span at the moment of artificial stimulation. Therefore, it is reasonable to expect that such activated oocytes will be less viable.

Although it is understandable that a sham-injected oocyte would require further treatment (e.g., exposure to calcium ionophore and DMAP) in order to be fully activated, it is unclear why the injected bovine sperm cell, itself, cannot stimulate the activation process as it would do during normal fertilisation. Bovine sperms have been shown to be more stably packaged than human, mouse, chinchilla and hamster sperm cells (Perreault et al., 1988). Table 2.3 present the result of oocyte activation in ICSI.

Higher blastocyst rates were obtained by a treatment using a combination of ionomycin and DMAP after ICSI compared with other activation treatments, however, no calves were produced after using this activation treatment (Rho et al., 1998b). It is important to examine both in vitro developments to blastocyst as well as calf production following their transfer, because oocyte activation treatments induce parthenogenetic development (Lie et al., 1999) and cause abnormal ploidy of embryos

Table 2.3: Bovine oocytes activation in ICSI

Piezo PN (86.3%), cleavage (71.8%), blastocyst (22.7%), ET (live born)

Horiuchi et al., 2002

Ethanol Cleavage (72%), blastocyst (20%), ET (live born, 5/10, 50%) No PN (88.9%), cleavage (58.5), blastocyst (7.3%),

ET (live born, 1/11, 9.1%)

Ionomycin Cleavage (50.7%), blastocyst (5.8%) Li et al., 2004 Ionomycin +

Piezo+ ethanol Cleavage (51%), blastocyst (14%)

Oikawa et al.,

Ethanol Cleavage (75.6%), blastocyst (39.4%), ET (live born, 9/19, 47.4%)

Kato and Nagao, 2009

PVP Cleavage (68.7%), blastocyst (20.9%)

(Rho et al., 1998a; Ock et al., 2003). Table 2.3 present the result of activation in ICSI bovine oocytes.