Introduction
World habitat destruction and pollution have lead to the development of strategies aimed at protecting and saving various species of plants and animals, including various canids. Unfortunately, animals continue to be killed by undesirable means, which include hunting, ecotourism, illegal pet trade and road accidents. With only a few inspectors to protect wildlife, biosecurity has become very fragile. Therefore, efforts to safeguard animal species that are living in areas with a severe risk to life preservation are needed.
Studies with in vitro fertilization of gametes, using the domestic dog as a model, are useful to expand knowledge in reproductive performance of various canids, as domestic and wild species share a common origin. All dogs are descended from the wolf and their reproductive cycles are nearly identical. In this way, in vitro procedures are supposed to serve to rehabilitate wildlife. Methods, such as in vitro embryo production, oocyte and embryo cryopreservation of canids, do offer the opportunity to recover infertile or subfertile populations. Other methods, such as short-term storage of sperm, might also be useful for successful in vitro procedures under conditions in which the female and male gametes are not synchronous and would be disposable after the claimed period for IVF.
In canine species, the establishment of in vitro-maturation protocols, with the aim to promote and maintain competence of the cumulus–oocyte complexes (COCs) for fertilization and cleavage, is still required. Despite the fact that various approaches (Cinone et al., Reference Cinone, Ghneim, Caira, A'quila and Minoia1992; Nickson et al., Reference Nickson, Boyd, Eckersall, Ferguson, Harvey and Renton1993; Srsen et al., Reference Srsen, Kalous, Nagyova, Sutovsky, King and Motlik1998; Hewitt & England, Reference Hewitt and England1999; Songsasen et al., Reference Songsasen, Yu and Leibo2002; Willingham-Rocky et al., Reference Willingham-Rocky, Hanna, Westhusin and Kraemer2002) have been tried in order to attempt in vitro meiosis of dog oocytes, acceptable rates of meiotic response for in vitro-matured bitch oocytes have not been obtained with most media. Major reasons point to difficulties in identifying which components are lacking in the medium, or which are acting as detrimental or suppressive compounds, or simply because the concentration of a substance needed to induce a response is unknown.
The objective of Experiment 1 (non-comparative experiment) in this study was to evaluate the developmental competence of in vitro-matured dog oocytes in three different media for in vitro fertilization with fresh ejaculated spermatozoa and cleavage. The objective of Experiment 2 (comparative experiment) was to compare the capability of oocytes, which had been matured in vitro in three different media and fertilized either with fresh or cooled extended homologous sperm, for cleavage and embryonic development. To our knowledge, a correlation between in vitro expansion of COCs and developmental competence of canine oocytes has not been reported in the literature and was also investigated. The results can serve as a useful guide to in vitro embryo production of domestic and wild canids.
Materials and methods
Oocyte collection and maturation
The experiments were performed using oocytes retrieved by slicing the ovaries of bitches at various unknown reproductive stages. Ovaries were collected at two centres for the control of carnivore populations: (1) Centro de Zoonoses – Estrada Bérico José Bernardes, 3489 – (Porto Alegre/RS; Brazil); (2) Associação Leopoldense de Proteção aos Animais (ALPA) – (São Leopoldo/RS, Brazil).
The mean age of 18 bitches used as ovary donors in Experiment 1 was 28.5 months (12–96) and in Experiment 2, the mean age of seven bitches used as ovary donors was 42.2 months (8–96).
Ovaries were transported to the laboratory at ambient temperature either in saline (0.9% NaCl) or phosphate-buffered saline (PBS). Mincing of ovaries was carried out in PBS supplemented with foetal calf serum (FCS) (0.5%). Maximal interval time from ovary collection to placement of oocytes in maturation medium was approximately 2 h. After slicing, the COCs with one or more layers of intact cumulus cells and dark regular and homogeneous cytoplasm (subjectively established as high quality oocytes) – Experiment 1: n = 253/1155 (21.9%); Experiment 2: n = 342/1106 (30.9%) – were randomly allocated into culture dishes containing four droplets with 100 μl of equilibrated IVM medium each with the following treatments: (A) TCM199 supplemented with 25 mM HEPES (v/v) (Sigma, M2520), 10% heat-inactivated estrous cow serum (ECS), 50 μg/ml gentamicin (Sigma, G1264), 2.2 mg/ml sodium bicarbonate (Sigma, S5761), 22 μg/ml pyruvic acid (Merk, 1.06619.0050), 20 μg/ml estradiol (Sigma, E 8875), 0.5 μg/ml FSH (Follitropin-V, Vetrepharm Inc.), 0.03 IU/ml HCG (Profasi HP) and 1 μg/ml human somatotropin (hST) (Lilly, FF1D44C) (control medium)]; (B) TCM199 supplemented with 25 mM HEPES/l (v/v) (Sigma, M2520), 10% heat-inactivated estrous cow serum (ECS) and 1 μg/ml human somatotropin (hST) (Lilly, FF1D44C); (C) TCM199 supplemented with 25 mM HEPES/l (v/v) (Sigma, M2520), 4 mg/ml polyvinylpyrrolidone (PVP) (Sigma, P0930) and 1 μg/ml human somatotropin (hST) (Lilly, FF1D44C).
Cumulus–oocyte complexes fertilized in vitro with fresh semen were previously matured for 48 h at 37 °C in a humidified atmosphere of 5% CO2 in air, while COCs fertilized in vitro with cooled semen were previously matured for 72 h at 37 °C in a humidified atmosphere of 5% CO2 in air. At the end of maturation period (48 h or 72 h), oocytes were evaluated for expansion on a four-point scale as previously reported (Ball et al., Reference Ball, Ax, First, Mahash, Muldoon, Saxana and Sadler1980). Briefly, complexes were rated as follows: 1 (compact, unexpanded complexes), 2 (outer cumulus cells slightly expanded), 3 (outer and inner cumulus cells slightly expanded) and 4 (fully expanded complexes). Oocyte maturation was followed by in vitro fertilization either with fresh or cooled extended homologous sperm and presumptive zygotes were cultured for up to 7 days.
Semen collection and spermatozoa assessing viability
A total of 12 ejaculates was used in Experiment 1. Semen was collected from a healthy known and in vitro fertility proven Whippet dog (aged 5 years). A total of six ejaculates from the same dog was used in Experiment 2. The samples were collected by digital manipulation in fractions (presecretion, sperm-rich and sperm-free fractions). Only the sperm-rich fraction was used for experiments. Sperm quality was observed by evaluating the sperm motility and the percentage of progressive motility, which were performed in all samples in Experiments 1 and 2 of this study. Sperm quality was assessed at all steps of semen preparation: following collection of spermatozoa, after washing by Percoll gradient and preceding IVF. Motility criteria followed the classification of Mies Filho (Reference Mies Filho1987). The percentage of total motile sperm (number of sperm with progressive motility/the total number of sperm observed) was assessed in 10 μl drops of sperm suspension, using a phase contrast microscope (×200).
Semen preservation
After collection, all ejaculates of sufficient quality were transported diluted (1:1) in Egg Yolk Tris extender (Günzel-Apel, Reference Günzel-Apel1994) at 26 °C in water bath to the laboratory.
In Experiment 2, the final volume, which was comprised by the ejaculate and extender, was divided into two samples, from which one half was used fresh to fertilize the oocytes matured for 48 h, while the second half was chilled and used afterwards to fertilize the oocytes matured for 72 h. The second half of the semen sample was placed in a 15 ml conical tube (Falcon #2095), and immersed in a 26 °C water bath inside a closed styrofoam box (17 × 22 × 14.5 cm). Sample was stored overnight at 4 °C in a refrigerator until IVF was carried out.
Sperm preparation
Aliquots of fresh and cooled diluted spermatozoa (∼1 ml) were pipetted into a 15 ml conical tube (Falcon # 2095) onto a two-layer discontinuous gradient formed by layering 1 ml of the 45% Percoll solution on top of 1 ml of 90% and centrifuged at 500 g for 20 min at room temperature. The supernatant was removed and the sperm pellet was washed with 5 ml of low bicarbonate–Tyrode's albumin lactate pyruvate (TALP) (Parrish et al., Reference Parrish, Susko-Parrish, Winner and First1988) by centrifugation at 500 g for an additional 10 min.
The pellet was then recovered after aspiration of the supernatant, and the spermatozoa were resuspended to give a final concentration of 2 × 106 cells/ml in Fert–TALP medium supplemented with 0.56 μg/ml heparin to induce capacitation (Rodrigues et al., Reference Rodrigues, Santos and Rodrigues2004). The cooled sperm samples were allowed to warm back up to room temperature before washing by Percoll.
In vitro fertilization (IVF) and in vitro culture (IVC) of oocytes
In Experiments 1 and 2, oocytes that had been previously matured either for 48 h or 72 h, were co-incubated at 37 °C with fresh or cooled sperm in a 5% CO2 in air atmosphere. At 24 h after insemination, the presumptive zygotes were partially denuded by repeated aspiration into a pipette and transferred to 70 μ1 drops of Synthetic Oviduct Fluid (SOF) (Tervitt et al., Reference Tervitt, Whittingham and Rowson1972) medium containing 4 mg/ml BSA, with Na pyruvate, glutamine, gentamicin and amino acids. Incubation was carried out under mineral oil in the same environmental conditions used for IVF, and COCs were cultured in vitro for up to 7 days.
Microscopy of embryos
Presumptive embryos completely dislodged from cumulus cells were evaluated subjectively with stereoscopic microscopy before being fixed and stained as described previously (Rodrigues et al., Reference Rodrigues, Santos and Rodrigues2004). Oocytes were mounted on slides, covered by coverslips, and afterwards fluorescence microscopy was performed to evaluate polyspermy, pronucleus formation, cleavage and embryonic development. Cleavage percentage was the per cent of total COCs that had more than one cell after fertilization. Polyspermy was the percentage of total COCs containing more than one decondensed sperm head, or one sperm head in the presence of more than one pronucleus, or more than two pronuclei (Kidson, Reference Kidson2004).
Statistical analysis
Data were evaluated by chi-squared analysis. Differences at the p < 0.05 level were considered significant. Experiment 1 was carried out using 12 replicates. Experiment 2 was carried out using six replicates.
Results
IVF Experiment 1 (non-comparative)
Final mean sperm motility of the 12 ejaculates was 71.66 ± 13.7% (± SEM) with a range of 50–95%. Sperm progressive velocity was 3.41% with a range of 3–5.
Seven embryos (2–8-cell stage) from a total of 253 fertilized oocytes (2.76%), were produced in this experiment, from which two achieved the 6–8-cell stage. These two embryos were derived from two out of 18 bitches used in the experiment. One 6–8-cell stage embryo was derived from a 2-year-old mixed-breed bitch, whose ovaries at the moment of retrieval showed no presence of follicles or corpora lutea at the surface. The other 6–8-cell embryo was derived from a 1-year-old Dackel bitch, whose ovaries at the moment of retrieval showed the presence of corpora lutea at the surface. The 6–8-cell stage embryos were from oocytes matured in medium A. However, cleavage rate did not differ among treatments (p = 0.277). The rates of pronucleus formation + cleavage are presented in Table 1, whereas the distribution of embryo development among treatments is presented in Table 2.
Table 1 Effect of different maturation media on IVF and cleavage of canine oocytes
ECS, estrous cow serum; PVP, polyvinylpyrrolidone; TCM, tissue cultured medium; others, immature/degenerated/unidentified.
Data from 12 replicates.
p ≥ 0.05.
Table 2 Embryo development of oocytes matured in vitro in three media
ECS, estrous cow serum; PVP, polyvinyl pyrrolidone; TCM, tissue culture medium; others, immature/degenerated/unidentified.
Data from 12 replicates.
IVF Experiment 2 (comparative)
Fresh spermatozoa exhibited motility and progressive velocity similar to that exhibited by cooled spermatozoa. Percentage sperm motility, in six ejaculates from a donor's dog, was on average 70.8% for fresh samples versus 57.5% for cooled sperm samples. Sperm progressive velocity was 3.6 for fresh samples versus 3.1 for cooled sperm samples.
Ten embryos (2–8-cell stage) from a total of 342 fertilized oocytes (2.92%), were produced in this experiment, from which three achieved the 6–8-cell stage. These embryos were derived from one 5-year-old mixed-breed bitch, whose ovaries at the moment of retrieval showed the presence of corpora lutea at the surface. Embryos developing to the 6–8-cell stage were derived from oocytes matured in medium A and fertilized with fresh semen (n = 2), and from one oocyte matured in medium C, previously fertilized with cooled semen (n = 1). The distribution of embryo development among treatment groups is presented in Table 4. No significantly differences were observed among treatments A, B and C, when analyzing pronucleus + cleavage rates (Table 3). Similarly to what was observed in Experiment 1, abnormal embryos were not seen in Experiment 2.
Table 3 Cleavage in vitro of canine oocytes matured in three different media and fertilized with fresh and cooled sperm
ECS, estrous cow serum; PVP, polyvinylpyrrolidone; TCM, tissue culture medium; others, immature/degenerated/unidentified.
Data from six replicates.
p ≥ 0.05.
Table 4 Embryo development of oocytes matured in vitro in three media and fertilized with fresh or cooled sperm
ECS, estrous cow serum; PVP, polyvinylpyrrolidone; TCM, tissue culture medium; others, immature/degenerated/unidentified.
Data from six replicates.
Polyspermy in Experiments 1 and 2
Failed membrane block (polyspermic fertilization) consisted mainly as decondensing sperm heads in the oocytes, except for a polyspermic oocyte with pronucleus distribution type II (two pronuclei apposed with one or more supplementary pronucleus or decondensed sperm heads located eccentrically) (Kidson, Reference Kidson2004) (Fig. 1).
Figure 1 Canine polyspermic embryo type II stained by Hoechst 33342 (left) and one immature oocyte (right) (×320) (Rodrigues, 2005).
In Experiments 1 and 2 lower, but not significant, frequencies of polyspermy were observed in oocytes submitted to treatment A. In Experiment 2, 9.61% (5/52) of oocytes fertilized with fresh ejaculated sperm, which were previously matured in medium A, were polyspermic compared with 22.22% oocytes matured in medium B (12/54) and in medium C (14/63), respectively. Nevertheless, oocytes fertilized with cooled sperm, were apparently the least prone to polyspermy in treatments A and C, when this parameter was compared with treatment B. The results for polyspermy in this study are presented in Tables 1 and 3.
Cumulus cell expansion in Experiments 1 and 2
In Experiment 1, expansion of cumulus cells in treatment A was similar to cumulus cells expansion observed in treatment B. In treatment A, 75% (9/12) of COCs and 50% (6/12) in treatment B showed cumulus cell expansion category 2, respectively. In treatment C, approximately 58% (7/12) of COCs showed this category of expansion, while 33% (4/12) of oocytes showed cumulus cell expansion category 1. Category 1 was not observed in COCs matured in media A and B. Frequency of oocytes showing category 3 in Experiment 1 was approximately 25% (3/12), 50% (6/12) and 8% (1/12) for media A, B and C respectively. None of the oocytes matured in the three media showed expansion of cumulus cells of category 4.
In Experiment 2, the most frequently seen cumulus expansion in oocytes matured for 48 h was for category 2. Frequencies were approximately 66% in treatments A (4/6) and B (4/6), and 50% (3/6) in treatment C, respectively.
The most frequently seen cumulus cells expansion for oocytes matured for 72 h was category 2 with frequencies of 50% in treatments A (3/6) and B (3/6), respectively. In treatment C, percentage of cumulus cells expansion category 2 was similar to cumulus cells expansion category 3. In treatment B, a single oocyte matured for 72 h was observed with cumulus cells expansion category 4. These data were not subjected to statistical analysis.
Discussion
In the bitch, the establishment of in vitro maturation protocols, with the aim to promote and maintain competence of the COCs for fertilization and cleavage is still required. Various protocols have been tried in order to attempt in vitro meiosis of dog oocytes (Cinone et al., Reference Cinone, Ghneim, Caira, A'quila and Minoia1992; Nickson et al., Reference Nickson, Boyd, Eckersall, Ferguson, Harvey and Renton1993; Srsen et al., Reference Srsen, Kalous, Nagyova, Sutovsky, King and Motlik1998; Hewitt & England, Reference Hewitt and England1999; Songsasen et al., Reference Songsasen, Yu and Leibo2002; Willingham-Rocky et al., Reference Willingham-Rocky, Hanna, Westhusin and Kraemer2002). Yet, meiotic response of in vitro-matured bitch oocytes is very unpredictable, because it is difficult to identify which components are lacking in the medium, or which are acting as detrimental or suppressive compounds, or simply because the concentration of a substance needed to induce a response is unknown. Also, current assays may not encompass all variables influencing cytoplasmic maturation.
It is generally accepted that the process of keeping the medium as simple as possible is the most reliable manner of knowing to what oocytes are being subjected. Despite the complexity, TCM199 has emerged as a commonly used medium for various species, including canids. Apparently, success of nuclear maturation to the metaphase II (MII) stage for canine oocytes matured in vitro is independent of gonadotropins, steroids, hormones such as somatotropin and protein supplements in the medium (Songsasen et al., Reference Songsasen, Yu and Leibo2002; dos Santos et al., Animal Reproduction, accepted). Nevertheless, it should be highlighted that maturation of COCs is probably the most critical in vitro step affecting subsequent embryo development to the blastocyst stage (Massip et al., Reference Massip, Mermillod, Van Langendonckt, Touze and Dessy1995). Many factors may influence the acquisition of oocyte competence during in vitro maturation, which can be assessed by the subsequent embryonic development. In vitro maturation is associated with migration of the cortical granules, whose contents are released upon sperm penetration and play a role in blocking polyspermy (Zheng & Sirard, Reference Zheng and Sirard1992).
Polyspermy is a generic term describing the penetration of two or more sperm into the oocyte, and can thus include dispermy, trispermy and tetraspermy or even greater degrees of multiple sperm entry. The pattern of polyspermy can be modulated by the maturational status of the oocyte (IVM conditions), as well as the dose and type of sperm used for IVF (Kidson, Reference Kidson2004).
McAvey et al. (Reference McAvey, Wortzman, Williams and Evans2002) reported that perturbation of the oocyte actin cytoskeleton or inadequacy of sperm-induced calcium signalling leads to increased polyspermy during IVF of mouse oocytes. Challenging oocytes with a higher number of sperm, as performed in this study, and/or suboptimal oocyte activation in response to sperm should not be disregarded as possible reasons for polyspermy in the present study. In both experiments, oocytes exposed to treatment A were apparently less prone to polyspermy, which could be the result of substances present in the medium and influenced by the addition of combined hormonal supplements. It might be possible that maturation of cumulus cells was enhanced in treatment A and therefore influenced supernumerary spermatozoa entry into the oocytes to a lesser degree. The rates of polyspermy observed in oocytes submitted to treatment A in this study were lower than the rates of polyspermy observed in a previous experiment, where the same medium was used to mature the oocytes (Rodrigues et al., Reference Rodrigues, Santos and Rodrigues2004). Polyspermy, and also other factors affecting embryo quality, could be the underlying cause of the low embryonic development we have observed in this study.
The action of endocrine factors that affect oocyte maturation in vitro may be quite different from the in vivo condition (Chian et al., Reference Chian, Lim and Tan2004). In vivo, there are four factors that are involved with oocyte developmental potential. These are normal growth phase progression, adequate follicle cell support during maturation, completion of intracellular programming before fertilization and the functioning of oocyte checkpoint surveillance mechanisms (Moor & Day, Reference Moor and Day2001).
Components in medium influence the levels of certain oocyte transcripts. Addition of serum to the maturation medium, such as estrous cow serum (ECS) inactivated by heat treatment, reduces the potentially damaging effects of competent proteins (Glied et al., Reference Glied, Rosenkrans Jr, Rorie, Munyon, Piersin, Miller and Rakes1996). Nevertheless, it has been suggested that the replacement of serum preparations by large inert molecules may reduce the potential risk of viral or prion contamination and variability associated with the previous sera (Chanson et al., Reference Chanson, Nocera, Senn, Grandi and Germond2001). One of these molecules is polyvinyl pyrrolidone (PVP), which has been used to exclude protein influence from culture medium in in vitro procedures in the bitch (Songsasen et al., Reference Songsasen, Yu and Leibo2002; Bolamba et al., Reference Bolamba, Russ, Olson, Sandler and Durrant2002). Indeed, culture serum-free systems allow researchers to examine more precisely oocyte requirements. However, serum has the effect of masking problems in the culture system. Moreover, it has been reported that serum-free medium results in zona pellucida hardening (Kito & Bavister, Reference Kito and Bavister1996). Nevertheless, a chemically defined IVM medium, such as TCM199 plus PVP, seems to supply canine oocytes requirements, supports both cleavage and development of normal embryos; this aspect was confirmed by the results in the present study. Regardless of that, it must be emphasized that oocytes were exposed to fetal bovine serum (FBS) during slicing of ovaries in the present study and, therefore, rates of cleavage observed in treatment C might have also resulted from temporary contact between oocytes and FBS, before transfer to culture medium. Rapid steroid actions, which can be stimulated by brief exposure to serum, are involved with a variety of cell signal transduction mechanisms (Chian et al., Reference Chian, Buckett and Tan2003). Assuming that the brief exposure to FBS might have contributed to oocyte priming, embryo development in treatment C was more likely a consequence of a directly or indirectly steroid-induced stimulation.
Information involving IVF of in vitro-matured dog oocytes is limited to a few papers (Yamada et al., Reference Yamada, Shimazu, Kawaji, Nakazawa, Naito and Toyoda1992; Metcalf, Reference Metcalfe1999; Otoi et al., Reference Otoi, Murakami, Fujii, Tanaka, Ooka, Une and Suzuki2000; England et al., Reference England, Verstegen and Hewitt2001; Songsasen et al., Reference Songsasen, Yu and Leibo2002, Rodrigues et al., Reference Rodrigues, Santos and Rodrigues2004). Current canine embryo culture systems are able to support in vitro-derived zygotes up to the 8-cell stage (Songsasen et al., Reference Songsasen, Yu and Leibo2002; Rodrigues et al., Reference Rodrigues, Santos and Rodrigues2004). In the majority of in vitro studies, dog zygotes do not progress to advanced stages (morula or blastocyst), with the exception of one report (Otoi et al., Reference Otoi, Murakami, Fujii, Tanaka, Ooka, Une and Suzuki2000).
Although culture medium supplemented with a physiological concentration of FSH or LH stimulates steroid (estradiol and progesterone) secretions from the cultured granulosa and cumulus cells (Chian et al., Reference Chian, Ao, Clarke, Tulandi and Tan1999), in vitro maturation studies examining the effect of exogenous steroid hormones on developmental competence of dog oocytes have described either negative (Rodrigues & Rodrigues, Reference Rodrigues and Rodrigues2003; Willingham-Rocky et al., Reference Willingham-Rocky, Hinrichs, Westhusin and Kraemer2003) or positive effects (Nickson et al., Reference Nickson, Boyd, Eckersall, Ferguson, Harvey and Renton1993; Wilingham & Kraemer, 2001).
It is still not known how supplements added to medium affect dog embryo development in vitro. For Experiments 1 and 2 in this study, embryos derived from in vitro-matured oocytes showed cleavage blockage around the 6–8-cell stage. Both incomplete maturation of cell cycle regulators and inadequate culture conditions may have been associated with the arrest of embryonic development and, in this study, poor embryonic competence may have originated at a nuclear and/or cytoplasmic level.
Similar to previous reports (Rodrigues et al., Reference Rodrigues, Santos and Rodrigues2004) and also in this study we observed a clearly variation between females for in vitro meiosis, cleavage and embryo development. Immature oocytes from two females in Experiment 1, and immature oocytes from one bitch in Experiment 2 underwent cleavage. Morphological appearance of donor's ovaries suggested that these three females were at a non-proliferative phase of estrous cycle at the time of ovary removal. Recently, it was demonstrated that larger follicles could exist in the dog cycle during diestrus (Songsassen & Wildt, 2005). Cha et al. (Reference Cha, Do and Chi1992) showed that human immature oocytes from the luteal phase had a significantly higher maturation rate than those obtained during the follicular phase. Interesting, these data support our previous (Reference Rodrigues, Santos and RodriguesRodrigues et al., 2004) and present findings, in which in vitro cleavage in the dog can be achieved regardless of the female reproductive stage. Nevertheless, the main problem in IVF studies in the bitch remains the limited number of donors, whose oocytes have maturational and developmental potential.
In various species (mouse, rat, cow and pig) oocytes produce an oocyte-secreted factor, termed the cumulus expansion-enabling factor (CEEF), which presumably plays an important role in formation and stabilization of the mucoid matrix molecules (Gilchrist et al., Reference Gilchrist, Ritter and Armstrong2004). Similarly to COC expansion within the follicle in vivo, COCs expand during in vitro maturation under the influence of gonadotropins (Yokoo et al., Reference Yokoo, Miyahayashi, Nagamura, Kimura, Sasada and Sato2002). Cumulus cell expansion, a parameter included in our evaluations in this study, requires a cAMP signal and this is readily induced by in vitro treatment with FSH. Luteinizing hormone (LH) may act directly upon cumulus cells from isolated COCs to stimulate synthesis of hyaluronan after FSH-mediated upregulation of sufficient numbers of suitable LH receptors on cumulus cells and oocyte membranes (Chen et al., Reference Chen, Russel and Larsen1994). The most vital influences on the oocyte are the cumulus cells, which are thought to be the source of a putative positive meiosis-inducing signal (Eppig, Reference Eppig2003, Reference Eppig2004) under the influence of gonadotropins. Furthermore, developmental competence of in vitro-matured oocytes has been reported as questionable when surrounding cumulus cells are removed (Chang et al., Reference Chang, Liu, Zhang, Grifo and Krey2005). Cumulus cell function and viability can be estimated by evaluating the degree of cumulus cell expansion, and this parameter is referred to as a gauge for approximating the COCs developmental potential (Kidson, Reference Kidson2004). Intriguingly, cumulus expansion seems to follow an unusual pattern in canids (Srsen et al., Reference Srsen, Kalous, Nagyova, Sutovsky, King and Motlik1998). We have previously observed that oocytes from ovaries of bitches at various cycle stages and, with large expanded and/or mucified cumulus cells after in vitro maturation, are not necessary the ones achieving meiosis. Furthermore, as it was not possible to establish a correlation between cleavage and COC expansion in this study, this parameter seems negligible and so far has not proved reliable in indicating whether an oocyte is developmental competent and if a successful fertilization and early embryonic development will be established.
It is noteworthy that four out of seven zygotes produced in Experiment 1 of this study arose from oocytes matured in medium A, which had FSH and LH added, while the remaining three arose from oocytes matured in medium B (n = 2) and medium C (n = 1). This observation suggests that gonadotropins and steroids seem to play a role in cytoplasmic maturation of canine oocytes rather than nuclear. Conversely, the addition of gonadotropins and steroids in medium was not sufficient to improve oocyte cleavage ability.
From the few zygotes produced, the highest percentage of embryos was arrested at the 2–5-cell stage (Fig. 2). It was previously hypothesized that arrested embryos enter a senescence like state and that the cell cycle regulatory protein p53 and the stress-related protein p66shc are responsible for this early embryonic arrest (Faveta et al., Reference Faveta, Robert, John, Betts and King2004).
Figure 2 Canine embryo (3–4-cell stage) derived from one oocyte matured in TCM199 supplemented with estrous cow serum (ECS) (×320) (Rodrigues, 2005).
It is noteworthy that the majority of embryos progressing to the 6–8-cell stage (Fig. 3) arose from oocytes matured in treatment A, except for one embryo produced in treatment C. Components in treatment A may have acted synergistically or cumulatively and contributed to embryo development.
Figure 3 Canine embryo (5–6-cell stage) derived from one oocyte matured in TCM199 supplemented with estrous cow serum (ECS) + estradiol + FSH + HCG (×320) (Rodrigues, 2005).
Mammalian embryos can be cultured successfully using both gas phase-containing atmospheric (20%) or reduced (5%) oxygen (O2) concentrations (Dumoulin et al., Reference Dumoulin, Meijers, Bras, Coonen, Geraedts and Evers1999), which are about 150 mmHg and 40 mmHg, respectively (Karagenc et al., Reference Karagenc, Sertkaya, Ciray, Ulug and Bahçeci2004). Dog embryos can develop in vitro up to the 8-cell stage under a high oxygen culture environment (Rodrigues et al., Reference Rodrigues, Santos and Rodrigues2004). However, it is known that the oxygen concentration within the reproductive tract of mammals is lower than in air (Fujitani et al., Reference Fujitani, Kasai, Ohtani, Nishimura, Yamada and Utsumi1997). As the concentration to which embryos are exposed in vivo varies between 2 and 8% in the oviduct and uterus, depending on species (Fischer & Bavister, Reference Fischer and Bavister1993), it is therefore advisable to culture embryos at low oxygen concentrations (5–7%) (Gardner & Lane, Reference Gardner, Lane, Gardner, Lane and Watson2004). Although the interpretation of these data requires some caution, we associate the possible toxic effects of high oxygen during the time of in vitro culture, and mainly represented by reactive oxygen species (ROS), among the variables that could have a negative impact on cleavage and embryo development in this study. Moreover, and as already reported for the mouse, it remains unknown if oxygen concentration is a stage-specific phenomenon on embryo development (Karagenc et al., Reference Karagenc, Sertkaya, Ciray, Ulug and Bahçeci2004).
Also, the high percentage of immature degenerated or unidentified oocytes found in this study, could be related to the yet inappropriate environment provided by the IVM/IVC systems. However, degeneration of oocytes as detected during IVM might be a normal process, because these are oocytes probably not representative of the selected population destined to ovulation (Villamediana et al., Reference Villamediana, Ruttlant, López-Bejar, Vidal and Paramio1999). Thus, degenerated or not well matured oocytes are more likely those incapable of successfully accomplishing their reproductive purpose, which is the competence to develop into embryos.
Another issue is the influence of sperm affecting in vitro cleavage. Cooling sperm decreases semen quality, and predisposes to altered patterns of sperm fertilization. From various criteria (viability, motility and functional ability of the sperm), the more frequently used for semen evaluation and for comparison between sperm preservation techniques is motility, as this is a clear sign of viability, and spermatozoa need to be motile to reach the oocyte and penetrate the ZP (Rota et al., Reference Rota, Furzi, Panzani and Camilo2004). Besides that, routine semen parameters may not reflect the quality of sperm DNA. Damage to sperm DNA includes ROS, to which spermatozoa are susceptible. This is because their plasma membranes contain large quantities of polyunsaturated fatty acids and their cytoplasm contains low concentrations of scavenging enzymes (Saleh & Agarwal, Reference Saleh and Agarwal2002).
We have previously examined the in vitro capacitation status of dog spermatozoa at the time of addition to the fertilization drops. We reported that at the start of IVF the mean number of spermatozoa capacitated and acrosome intact (B pattern), and capacitated and acrosome reacted (AR pattern) was measured to be 51.47 ± 6.24, and 20.64 ± 5.02, respectively. At that time, we observed a normal cleavage rate of 10.1% of bitch oocytes in association with these numbers (Rodrigues et al., Reference Rodrigues, Santos and Rodrigues2004).
In this study, IVF of oocytes with cooled sperm was equally efficient for cleavage, when compared with IVF of oocytes with fresh semen. Therefore, the suboptimal fertilization results obtained in the present study are less likely to be related to the sperm type, i.e., fresh or cooled, and should thus be rather attributed to other cell changes induced during sperm processing, for example inadequate capacitation of spermatozoa, although this statement deserves in-depth investigation and remains to be proven.
Sperm susceptibility to cooling was observed in a few samples from the donor dog. This aspect was demonstrated by the reduction in motility after spermatozoa back rewarming. In Experiment 2, the postrewarming score of cooled semen samples showed that cleavage was achieved even when sperm motility was as low as 40%. Despite the fact that higher percentages of sperm motility are an effective predictor of in vitro fertilization potential, samples with reduced numbers of motile spermatozoa could still retain most intrinsic characteristics required for fertilize an oocyte. In Experiment 2, cooled sperm was proved to fertilize oocytes, as assayed by cleavage, and therefore this suggests that both cell motility and membrane integrity were preserved in the sperm population. The negative effects of storage have apparently been overcome by maintenance of sperm in Tris–egg yolk diluent, which is known to stabilize the sperm membrane due to the protective effect of egg yolk. In vitro preservation of fertility on cooled dog semen in Experiment 2 is encouraging because cooled sperm could be useful in IVF studies in canine species, especially under circumstances where male and female gametes are not synchronous disposable at the moment of insemination.
In conclusion, the results in this study indicate that competence of oocytes matured in vitro in TCM199, is independent of medium supplements, as assessed by the subsequent cleavage. The results here suggest that the kind of supplement added to the maturation medium is not involved in the acquisition of competence of in vitro-matured canine oocytes. Also, it was demonstrated for the first time that in vitro-matured canine oocytes could be fertilized by homologous cooled spermatozoa and progress to cleavage, even when at highly compromised rates. Lastly, it was concluded that canine oocytes cleave in vitro regardless the extension of COC expansion.
The presented data reinforces our thoughts that a prepattern of cytoplasmic attributes are responsible for oocyte developmental capacity. Intrafollicular ovarian dynamics coupled to the inherent quality of the oocyte have yet to meet the appropriate in vitro culture environment, where the in vivo situation could be mimicked. The identification of specific means by which the developmental potential of canine oocytes can be enhanced remains as the great challenge of the in vitro programme in the bitch.
Acknowledgements
The authors thank the Centro de Zoonoses and ALPA for generously providing bitch ovaries. We are also grateful to Prof. Carlos Bacelar (in memoriam) and his wife Mrs Eliete Bacelar (Infinitu's Kennel, Porto Alegre/RS, Brazil) for his kind cooperation and valuable help in supplying the stud dog (Enigma).
