Introduction
Rabbits are used frequently for studies in reproductive biotechnology and genetic modelling. Thus, oocyte recovery is essential for a wide variety of assisted reproductive technologies and associated ovarian stimulation protocols are usually used to increase the number of oocytes collected. In rabbits, superovulation protocols have been performed widely with equine chorionic gonadotropin (eCG) or follicle stimulating hormone (FSH) (Kauffman et al., Reference Kauffman, Schmidt, Rall and Hoeg1998; Mehaisen et al., Reference Mehaisen, Vicente, Lavara and Viudes-de-Castro2005; Salvetti et al., Reference Salvetti, Theau-Clement, Beckers, Hurtaud, Guerin, Neto, Falieres and Joly2007; Cortell et al., Reference Cortell, Vicente, Mocé, Marco-Jiménez and Viudes-de-Castro2008; Viudes-de-Castro et al., Reference Viudes-de-Castro, Cortell, Mocé, Marco-Jiménez, Joly and Vicente2009). Nevertheless, the recovery efficiency and oocyte quality of these superovulation treatments are both highly variable. One of the causes of the variability in the response after gonadotrophin stimulation may be related to the administration protocol: whereas a single eCG injection is enough to induce superovulation, FSH, due to it shorter lifespan, must be administered at least twice daily for 3 days (Rose et al. Reference Rose, Gaines Das and Balen2000). However, in order to limit animal manipulation, superovulation treatments with FSH diluted in polyvinylpyrrolidone (PVP) solutions have been tested and have resulted in a good superovulation response in distinct species, including rabbits (Kanayama et al., Reference Kanayama, Sankai, Nariai, Endo and Sakuma1994; Hashimoto et al. Reference Hashimoto, Kuramochi, Aoyagi, Takahashi, Ueda, Hirao, Kamei, Kitada and Hirasawa2004; Mehaisen et al., Reference Mehaisen, Viudes de Castro, Vicente and Lavara2006; Cortell et al., Reference Cortell, Vicente, Mocé, Marco-Jiménez and Viudes-de-Castro2008; Viudes-de-Castro et al., Reference Viudes-de-Castro, Cortell, Mocé, Marco-Jiménez, Joly and Vicente2009).
In addition, when using FSH isolated from the pituitary gland, the ratio between the luteinising hormone (LH) and FSH is unknown, which may results in a variable response to superovulation treatments. In rabbits, the effect of LH on superovulation has been studied using both porcine-purified FSH (Hashimoto et al., Reference Hashimoto, Kuramochi, Aoyagi, Takahashi, Ueda, Hirao, Kamei, Kitada and Hirasawa2004; Salvetti et al., Reference Salvetti, Theau-Clement, Beckers, Hurtaud, Guerin, Neto, Falieres and Joly2007) and human recombinant FSH (Cortell et al., Reference Cortell, Vicente, Mocé, Marco-Jiménez and Viudes-de-Castro2008; Viudes-de-Castro et al., Reference Viudes-de-Castro, Cortell, Mocé, Marco-Jiménez, Joly and Vicente2009). In all cases, a superovulatory response has been achieved, however it would be very useful to determine how oocyte quality may be affected by these superovulation procedures.
Oocyte quality determination is critical for most human and animal assisted reproductive technologies. Detrimental effects of superovulation on oocyte quality are often observed (Moor et al., Reference Moor, Osborn and Crosby1985; Blondin et al., Reference Blondin, Coenen, Guilbault and Sirard1996; Ertzeid and Storeng, Reference Ertzeid and Storeng2001; Van der Auwera and D'Hooghe, Reference Van der Auwera and D'Hooghe2001). Developmental competence is acquired during folliculogenesis as the oocyte grows and during the period of oocyte maturation (Rodrigues et al., Reference Rodrigues, Limback, McGinnis, Plancha and Albertini2008). It is probable that alterations in oocyte quality could be related to a failure in its maturation process. It is well known that organelle and chromosome redistribution are necessary steps in oocyte maturation and meiosis resumption (Ferreira et al., Reference Ferreira, Vireque, Adona, Meirelles, Ferriani and Navarro2009), indeed metabolic, structural and numerical defects have been associated with pre-ovulatory maturational failure for the oocyte and premature arrest or abnormal development for the embryo (Van Blerkom et al., Reference Van Blerkom, Davis and Lee1995). While it seems that oxidative metabolism is the primary source of energy production during oocytes maturation (Krisher, Reference Krisher2004), mitochondria, by means of their ability to generate ATP, have a central role in the normality of early mammalian development. In order to evaluate embryo and oocytes competence, the ATP concentration and mitochondrial membrane potential in oocytes has been widely studied in different species, showing a high correlation between oocyte ATP concentration and developmental competence of the resulting embryo (Van Blerkom et al., Reference Van Blerkom, Davis and Lee1995; Stojkovic et al., Reference Stojkovic, Machado, Stojkovic, Zakhartchenko, Hutzler, Gonçalves and Wolf2001; Cummins, Reference Cummins2002; Tamassia et al., Reference Tamassia, Nuttinck, May-Panloup, Reynier, Heyman, Charpigny, Stojkovic, Hiendleder, Renard and Chastant-Maillard2004; Van Blerkom, Reference Van Blerkom2004).
The aim of this study was to assess oocyte ATP content to evaluate the effect of FSH origin and administration protocols on oocyte quality.
Materials and methods
The research was carried out at the experimental farm of the Centro de Investigación y Tecnología Animal (Segorbe, Castellón, Spain). The experiments were approved by the Ethics Committee of the Instituto Valenciano de Investigaciones Agrarias.
All the chemicals unless otherwise stated were reagent grade and purchased from Sigma-Aldrich Química S.A. (Alcobendas, Madrid, Spain). Other experiments were carried out in the Animal Technology Center (CITA-IVIA, Spain).
Animals
Sexually mature White New Zealand rabbit does and males were housed in flat deck cages with light alternating on a cycle of 16 h light and 8 h dark; they were fed with the same commercial diet (17.5% crude protein, 2.3% ether extract, 16.8% crude fibre, 2600 kcal DE/kg) and had free access to water.
Superovulation treatment and ovulation induction
In the first phase of this study, 73 rabbit does were randomly distributed into four groups:
• control group: non-superovulated, females were injected every 24 h for 3 days, with 0.4 ml of 30% polyvinylpyrrolidone (PVP; molecular weight, 40000) in distilled water;
• rhFSH3 group: females were injected, every 24 h for 3 days, with 0.6 μg of recombinant human FSH (Gonal-F® 75, Serono, Italy) diluted in 0.4 ml of 30% PVP;
• pFSH3 group: females were injected every 24 h for 3 days with 11.4 μg of porcine FSH (Stimufol®, Reprobiol, Belgium) diluted in 0.4 ml of 30% PVP;
• pFSH5 group: after dissolving the hormones in physiological saline to a final concentration of 18 μg/ml of porcine FSH (Stimufol®, Reprobiol, Belgium), females were injected twice (12 h intervals) 0.25 ml on day 1, twice 0.5 ml on day 2 and only one injection of 0.25 ml on the morning of day 3.
All females were induced to ovulate, 24 h after the last injection, with 2 μg of Buserelin acetate (Suprefact, Hoechst).
Oocyte recovery
Does were euthanized 14 h after ovulation induction and the reproductive tract was immediately removed. Oocytes were recovered by perfusion of each oviduct with Dulbecco's phosphate-buffered saline (DPBS). Collected liquid was poured into Petri dishes and scored under a stereomicroscope. Cumulus–oocytes complexes (COCs) were localized and only those with homogeneous cytoplasm and compacted cumulus were selected to perform the ATP analysis.
The number of follicles with scare ovulation was recorded to estimated the ovulation rate and number of recovered and normal oocytes were recorded by female.
ATP measurement
Selected COCs were suspended in 0.5 % of hyaluronidase in DPBS in order to remove cumulus cells. After mechanical removal, the oocytes from each females were distributed randomly into three groups: (i) in the first group (control group), ATP was measured immediately after recovering fresh oocytes at room temperature (25ºC); (ii) in the second group, ATP was measured after culture of oocytes for 2 h at 25ºC (25ºC – 2 h group); and (iii) finally, in the third group, ATP measurements were done after culture of oocytes for 2 h at 38.5ºC and in 5% CO2 in air (38.5ºC – 2 h group).
Concentration of ATP was measured using a commercial assay kit based on the luciferin-luciferase reaction (Bioluminescent Somatic Cell Assay Kit, Fl-ASC; Sigma) following the technique described by Rieger (Reference Rieger1997) and the manufacturer's recommendations. Briefly, two oocytes in 50 μl of PBS were transferred into 5 ml plastic tubes equilibrated on ice (0ºC). One hundred microlitres of ice-cold somatic cell reagent (FL-SAR reagent) were then added to the oocytes and incubated for 5 min on ice. Next, 100 μl of diluted ice-cold assay mix (FL-AAM reagent; 1:25 with ATP assay mix dilution buffer, FL-AAB reagent) was added, and the tubes were kept for an additional 5 min at room temperature in the dark. The solution was then transferred into appropriate plastic tubes fitted for the high-sensitivity (0.01 pmol) luminometer (Bioluminat Junior; Berthold, Wildbad, Germany) and luminescence was measured. To obtain simultaneous measurements of samples, a set of tubes with oocytes was measured once and again in reverse order. The mean of these two values expressed in Relative Light Unit was calculated to establish our final value. A seven-point standard curve (0–6 pmol/tube) was determined and, after every 20 oocytes, a negative control was run. The ATP content was calculated using the formula derived from the linear regression of the standard curve.
Evaluation of developmental competence
As the pFSH5 group was the only one that showed significant differences in ATP content relative to the superovulation group, in the second phase of the study developmental competence of oocytes from the control and pFSH5 superovulation groups was examined.
Twenty-four does were inseminated and ovulation was induced with 2 μg buserelin acetate (Suprefact; Hoechst Marion Roussel, S.A., Madrid, Spain) given intramuscularly 24 h after the last injection.
Presumptive zygotes were collected 14 to 16 h after artificial insemination. In total, 351 presumptive zygotes (155 control and 196 superovulated) were washed twice in DPBS with 2 g BSA/l and placed into culture dishes in 1 ml of HAMS F12 supplemented with 20% fetal calf serum. The cultures were maintained for 78 h at 38.5ºC in humidified 5% CO2 in air and the development to blastocyst was recorded.
Statistical analyses
The effect of superovulation treatment on the ovulation rate and recovered oocytes were analysed by a General Linear Model Statgraphics®Plus5.1 (Statistical Graphics Corp., Rockville, MO, USA). Females that did not ovulate were eliminated from study.
The effect of superovulation treatment (control, rhFSH3, pFSH3 or pFSH5), the culture, the temperature and its interaction (0 h, 2 h – 25ºC and 2 h – 38.5ºC groups) were used as fixed effects on the oocytes ATP content. These effects were analysed by a General Linear Model Statgraphics®Plus5.1 (Statistical Graphics Corp., Rockville, MO, USA).
The effect of superovulation (control and pFSH5 groups) on the developmental competence was compared using the General Linear Mixed Model (GLIMMIX) procedure of the Statistical Analysis System (SAS Inc. Cary, NC, USA). Treatments (pFSH5 superovulation or control groups) were considered fixed effects in the model, while superovulation session and culture session were regarded as random effects. A binomial distribution with a logit-link function was used. Means were separated using Fisher's protected least significant difference (LSD), with treatment effect declared significant at P < 0.05. For ease of interpretation, means and standard errors shown in Table 3 are presented on the scale of the data (logit scale).
Results
In the first phase of the study 73 does were treated, five females were rejected for failure in ovulation induction. The ovulation rate and the number of recovered oocytes in the pFSH5 group were significantly higher than the other groups (P < 0.05, Table 1); while the rhFSH3 group showed ovulation rate and number of recovered oocytes significantly higher than the control group, the pFSH3 group results were similar to the control and rhFSH3 groups.
Effect of superovulation treatment on ovulation rate and number of oocytes recovered (least square means ± standard error).
Control: females treated every 24 h for 3 days, with 0.4 ml of 30% polyvinylpyrrolidone (PVP).
pFSH3 group: females treated every 24 h for 3 days with 11.4 μg of porcine follicle stimulating hormone (FSH) diluted in 0.4 ml of 30% PVP.
rhFSH3: females treated, every 24 h for 3 days, with 0.6 μg of human recombinant FSH diluted in 0.4 ml of 30% PVP.
pFSH5 group: females treated twice (12 h intervals) with 4.5 μg on day 1, twice 9.0 μg on day 2 and only one injection of 4.5 μg on the morning of day 3 of porcine FSH dissolved in physiological saline.
a,b,cValues with different superscripts in the same column are statistically different (P < 0.05).
Conversely, as shown in Table 2, differences among superovulation treatments were found in ATP levels. In the pFSH5 group, the ATP level was significantly lower than in other groups (5.63 ± 0.14 pmol of ATP for the pFSH group versus 6.42 ± 0.13 and 6.19 ± 0.15 pmol of ATP for rhFSH3 and pFSH3, respectively; P < 0.05) but similar to the control group.
Effect of superovulation treatment and culture temperature on ATP content (least square means ± standard error).
n: number of oocytes.
Control: females treated every 24 h for 3 days, with 0.4 ml of 30% polyvinylpyrrolidone (PVP).
pFSH3 group: females treated every 24 h for 3 days with 11.4 μg of porcine follicle stimulating hormone (FSH) diluted in 0.4 ml of 30% PVP.
rhFSH3: females treated, every 24 h for 3 days, with 0.6 μg of human recombinant FSH diluted in 0.4 ml of 30% PVP.
pFSH5 group: females treated twice (12 h intervals) with 4.5 μg on day 1, twice 9.0 μg on day 2 and only one injection of 4.5 μg on the morning of day 3 of porcine FSH dissolved in physiological saline.
a,b,cValues with different superscripts in the same column are statistically different (P < 0.05).
Interaction was not significant.
Regarding the culture effect on ATP contents, oocytes cultured for 2 h at 25ºC or 38.5ºC presented higher ATP levels than those measured immediately after recovery (6.17 ± 0.16 and 7.08 ± 0.13 pmol of ATP for 25ºC – 2 h and 38.5ºC – 2 h groups respectively, versus 4.95 ± 0.10 pmol of ATP for the control group; P < 0.05, Table 2). In addition, ATP levels in the 38.5ºC cultured oocytes group were significantly higher than in the 25ºC cultured oocytes group.
For the second phase of the study, the developmental competence of pFSH5 superovulated oocytes and control groups is shown in Table 3. The pFSH5 superovulation treatment has a substantial influence on the developmental ability of oocytes, with 24.28% of ova developing into hatched blastocysts after in vivo fertilization versus a figure of 80.39% for the control group (P < 0.0001). Considering only the fertilized ova, the developing rate for the pFSH5 group was significantly lower than that of the control group (79.04% versus 95.16% for the pFSH5 and control groups, respectively).
Effect of pFSH5 superovulation treatment on developmental competence (mean ± standard error).
n: number of presumptive zygotes.
HR: hatching rate = (hatched blastocyst/cultured presumptive zygotes) × 100.
DR: development Rate = ((hatched blastocyst – oocytes)/cultured embryos) × 100.
a,bValues with different superscripts in the same column are statistically different (P < 0.05).
Discussion
Several studies have revealed that administration of exogenous gonadotropin yields high numbers of oocytes and embryos but may result in a decrease in its developmental competence (Fujimoto et al., Reference Fujimoto, Pahlavan and Dukelow1974; Moor et al., Reference Moor, Osborn and Crosby1985; Blondin et al., Reference Blondin, Coenen, Guilbault and Sirard1996; Kauffman et al., Reference Kauffman, Schmidt, Rall and Hoeg1998; Ertzeid & Storeng, Reference Ertzeid and Storeng2001; Mehaisen et al., Reference Mehaisen, Vicente, Lavara and Viudes-de-Castro2005). Nevertheless, the way in which gametes are altered by gonadotropins could be a response to different factors, including an alteration in oocyte maturation or in its energy metabolism. Mitochondria, by means of their ability to generate ATP by oxidative phosphorylation, are the main organelle involved in energy regulation (Ramalho-Santos et al., Reference Ramalho-Santos, Varum, Amaral, Mota, Sousa and Amaral2009).
Given that it has been observed that FSH in the absence of LH could induce a superovulatory response in rabbit does using both purified porcine FSH or recombinant human FSH (Salvetti et al., Reference Salvetti, Theau-Clement, Beckers, Hurtaud, Guerin, Neto, Falieres and Joly2007; Cortell et al., Reference Cortell, Vicente, Mocé, Marco-Jiménez and Viudes-de-Castro2008; Viudes-de-Castro et al., Reference Viudes-de-Castro, Cortell, Mocé, Marco-Jiménez, Joly and Vicente2009), in the present study the effect of these hormonal preparations on ovulation rate and oocyte quality was compared. Regarding the superovulatory response, both purified pFSH administered in five injections, twice a day (pFSH5), and rhFSH dissolved in PVP administered in three injections, once a day for 3 days (rhFSH3), resulted in a higher ovulation rate than the control group. However, in spite of their ability to induce a superovulation response, while rhFSH3 reached similar results to those reported previously with oFSH, pFSH and rhFSH, increasing to twice the ovulation rate and the number of oocytes or embryos recovered in superovulated does (Kauffman et al., Reference Kauffman, Schmidt, Rall and Hoeg1998; Mehaisen et al., Reference Mehaisen, Viudes de Castro, Vicente and Lavara2006; Salvetti et al., Reference Salvetti, Theau-Clement, Beckers, Hurtaud, Guerin, Neto, Falieres and Joly2007; Viudes-de-Castro et al., Reference Viudes-de-Castro, Cortell, Mocé, Marco-Jiménez, Joly and Vicente2009), the pFSH5 treatment showed a ovulation rate 3.5 times higher than that of the control group. Despite an increase in the number of oocytes produced per female, when animals were treated with the pFSH5 protocol the blastocyst developmental rate per oocyte was significantly decreased compared with control females (15% less development rate). Superovulation treatments with FSH diluted in PVP permits slower absorption of FSH and more prolonged blood concentration levels. Conversely, when pFSH was administered once a day for 3 days, no superovulatory response was observed. The poorer ovarian response in the pFSH3 group at the concentration used in the present work may be affected by the treatment schedule or by the hormone vehicle. This idea would be supported by our previous studies (data not shown) in which rhFSH applied in saline serum twice a day for 5 days resulted in a higher ovulation rate than if applied in PVP once a day for 3 days. Thus, these results may indicate that the amount of FSH reaching the ovary with the dose of pFSH used in the present work is less when the hormone is administered diluted in PVP than when it is administered diluted in saline serum. Although PVP has been used as a hormone vehicle previously successfully in rabbit does (Kanayama et al., Reference Kanayama, Endo and Sakuma1992; Hashimoto et al., Reference Hashimoto, Kuramochi, Aoyagi, Takahashi, Ueda, Hirao, Kamei, Kitada and Hirasawa2004; Cortell et al., Reference Cortell, Vicente, Mocé, Marco-Jiménez and Viudes-de-Castro2008; Viudes-de-Castro et al., Reference Viudes-de-Castro, Cortell, Mocé, Marco-Jiménez, Joly and Vicente2009), to our knowledge this regime has not been compared with the same hormonal dose administrated in saline serum. However, it has been noted that the reduction of injection number using PVP as vehicle may affect the ovarian response after superovulation (D'Alessandro et al., Reference D'Alessandro, Martemucci, Colonna, Borghese, Terzano and Bellitti2001; Lopes da Costa et al., Reference Lopes da Costa, Chagas e Silva and Robalo Silva2001). In order to determine the effect of these superovulation treatments on oocyte quality, the amount of ATP in recovered oocytes was determined. It has been described previously that mitochondrial dysfunction and ATP content may be related to developmental competence in oocytes and embryos (Van Blerkom et al., Reference Van Blerkom, Davis and Lee1995; Thouas et al., Reference Thouas, Trounson, Wolvetang and Jones2004; Nagano et al., Reference Nagano, Katagiri and Takahashi2006). A significant difference in ATP content between different superovulatory treatments was found. Our data suggest that the reduced level of ATP in pFSH5-produced oocytes may reflect a minor mitochondrial activity, which plays a critical role in the acquisition of developmental competence in the oocyte. It is well known that the oocyte maturation process depends on FSH and LH stimulation and oocyte–follicle interaction (Hillier, Reference Hillier2009) and superovulation treatments could affect granulosa cell activity or accelerate follicular growth (Blondin et al., Reference Blondin, Coenen, Guilbault and Sirard1996). In fact, an effect on ATP content by superovulation treatment has been described previously both in mouse (Combelles and Albertini, Reference Combelles and Albertini2003) and hamster (Lee et al., Reference Lee, Oh, Lee, Han and Lim2006).
However, how ATP levels are affected during oocyte maturation is still unclear and seems to be very species specific; it has been observed that in vitro maturation and maturation conditions may effect mitochondrial activity in rabbits (Kanaya et al., Reference Kanaya, Hashimoto, Teramura, Morimoto, Matsumoto, Saeki, Iritani and Hosoi2007), mouse (Combelles and Albertini, Reference Combelles and Albertini2003), cows (Stojkovic et al., Reference Stojkovic, Machado, Stojkovic, Zakhartchenko, Hutzler, Gonçalves and Wolf2001; Nagano et al., Reference Nagano, Katagiri and Takahashi2006), gilts (Brevini et al., Reference Brevini, Vassena, Francisci and Gandolfi2005) or cats (Freistedt et al., Reference Freistedt, Stojkovic, Wolf and Stojkovic2001).
Conversely, oocyte ATP levels increased significantly after culturing for 2 h, however results were higher in those oocytes cultured at 38.5ºC than in those cultured at 25ºC. This ATP variation with the incubation temperature suggests that incubation at 38.5ºC might allow the oocytes to recover ATP synthesis more quickly than at 25ºC. The low ATP contents when oocytes were recovered may due to ATP release under stress conditions and its degradation by ecto-ATPases sited in cell surface (Joseph et al., Reference Joseph, Buchakjian and Dubyak2003; Schwiebert and Zsembery, Reference Schwiebert and Zsembery2003). In fact, this scenario would be in accordance with observations by Tamassia et al. (Reference Tamassia, Nuttinck, May-Panloup, Reynier, Heyman, Charpigny, Stojkovic, Hiendleder, Renard and Chastant-Maillard2004) in cows, who detected lower ATP levels in oocytes obtained from the slaughterhouse than in oocytes obtained from ovum pick-up (OPU), probably due to the more stressful conditions and transport when they were recovered from a sacrificed female. Moreover, these oocytes achieved ATP contents similar to those of oocytes from OPU after culture.
Finally, our results show that porcine FSH administered in five doses, every 12 h, may induce a greater response in ovulation rate than recombinant human FSH dissolved in PVP and administered in three doses, every 24 h. Conversely, a negative effect on oocyte quality has been observed in the pFSH5 group in ATP production when compared with other superovulation treatments. Possibly, the alteration in oocyte quality affected the competence of oocyte to be fertilized and could explain the lower development rate observed. This finding could be related to a failure in its maturation process probably due to the long period between the last FSH dose and ovulation induction. This situation suggests that, in the case of FSH in saline adjuvant, ovulation induction time may affect the developmental competence of oocytes.
Findings from the current study suggest that rhFSH dissolved in PVP administered once a day for 3 days allows the achievement of adequate follicular maturation during ovarian stimulation. By contrast, in the case of pFSH5, the long period between the last FSH dose and ovulation induction could affect the developmental competence of oocytes.
In conclusion, the reduced level of ATP may reflect reduced activity of mitochondria, which play a critical role for acquisition of developmental competence.
Acknowledgements
This research was supported in part by the project RTA2010–00117–00–00 from INIA and European FEDER Funds.
