Superovulation Induced by Multiple-Dose Administration of Pregnant Mare Serum Gonadotropin in Bitches

Superovulation is the most common technique used in assisted reproduction, and it is important for improving the efficiency of oocyte and animal production and reducing the number of oocyte donors (Takeo et al., 2019). Canine reproductive physiology has unique characteristics that make extrapolation from farm animals (horses, cows, sheep, goats, pigs) unsuccessful in this species (Kutzler, 2018). Domestic bitches are non-seasonal monoestrus; they ovulate only once or twice per year with a few exceptions (Fuller). In most mammals, oocyte maturation occurs within the ovarian follicle, and mature oocytes are ovulated and fertilized within the oviduct. However, canine oocytes are ovulated at an immature state that requires a further 48 to 72 h for completion of meiosis within the oviduct (Chastant-Maillard et al., 2011; Reynaud et al., 2012). Because of this unique reproductive physiological characteristic and the paucity of information concerning factors regulating postovulatory oocyte maturation, superovulation techniques for dogs are not well established (Lee et al., 2017).

Canine embryos are scarce biological material, because of the inefficiency of superovulation and cycle induction/synchronization protocols (Rodrigues and Rodrigues, 2010). Difficulties encountered in collecting in vivo produced embryos and current the impossibility to produce canine embryos in vitro are other limiting factors. Currently, in vitro oocytes surgically obtained from female dogs in natural rut (Lee et al., 2005). However, as dogs are monoesters, and have estrous cycle of ~6 months, information on the duration of estrus is usually missing (Nakao et al., 1985). Following natural estrus, without superovulation, an average of only six to eight good-quality embryos can be collected from each female dog (Chastant-Maillard et al., 2010). For livestock, such as cattle (Bó and Mapletoft, 2014), pigs (Angel et al., 2014) and sheep (Figueira et al., 2020), the superovulation method is well established. However, because the reproductive biology of dogs is unique, the superovulation method is not yet well established for this species (Hirata et al., 2018). Consequently, the availability of canine oocytes and knowledge on the reproductive physiology of bitches remain severely limited.

In this study, our aim is to collect mature oocytes by continuous injection of PMSG to superovulation the bitches. These findings may contribute to basic technologies such as in vitro maturation, in-vitro fertilization, and somatic cell nuclear transfer that are used in studies on reproductive aspects of dogs.

Three mixed-breed bitches aged 1.5 years and weighing 5 kg in anestrus were used in the study. They were provided by the Animal Hospital of The College of Agriculture, Yanbian University. The bitches were confirmed to be no abnormity of the reproductive system by ultrasonography and no symptom of estrus by vaginal smears and vulvar observation. They were fed standard commercial dog food once a day and given water ad libitum. Through vaginal cytological examination and vulvar observation, it was confirmed that the bitch was in a state of anestrus. 

All bitches were administered 200 IU pregnant mare serum gonadotropin (PMSG, Deasung Microbiological Labs. Co, Ltd, Seoul, South Korea) intramuscularly for 8 consecutive days, followed by a 3-day interval. Then on the 12th day, they received intramuscular injection of human chorionic gonadotropin (hCG; Deasung Microbiological Labs. Co, Ltd, Seoul, South Korea) at a dosage of 2000 IU (Fig. 1). During the entire trial, all animals were examined vaginal cytology and ultrasonic testing every day. After laparotomy, the dogs were sent back to the Animal Hospital. None of the dogs were used repeatedly. The first day of treatment was considered as day 1 of the experiment.

After the superovulation treatment, the preoestrus and estrus stages, as well as the expected ovulation period, are determined by sexual behavior records, vaginal cytology and related evaluation criteria are based on those by Jurczak et al. (2016). Observe the normal ovary in anestrus and the follicles and corpora lutea by (England and Yeager, 1993).

The dogs were fasted for 1 day before the experiment. All surgeries were performed under general anesthesia. Atropine sulfate (0.05 mg/kg; ShengDa, China) and acepromazine maleate (0.025 mg/kg; ShengDa, China) were administered as a pretreatment, and ketamine (5 mg/kg; ShengDa, China) was administered to induce anesthesia. Anesthesia was maintained using isoflurane. The laparotomy was performed on the 23rd day of the experiment.

An approximately 5 cm incision was made at 2 cm of the midline above the dogs umbilicus. The skin and subcutaneous tissue were cut to expose the abdominal cavity. A bending hemostatic forceps was used to extend the incision backward along the abdominal wall to 2 to-3 cm from the kidney, hook the uterine angle on one side, pull toward the incision, find and remove the ovarian suspension ligament, and then remove the ovary from the abdominal cavity. The same procedure was performed on the other ovary. The removed uterus was quickly placed in a vacuum flask and taken to the laboratory. Finally, the abdominal incision was closed using a two-layer closure followed by a surgical adhesive applied along the skin incision. Standard post-operative care of the bitches was performed by veterinarians.

After removal of the uterus, the entire bursal compartment around an ovary, including a small portion of the uterine horn immediately distal to the uterotubal junction, was removed. the excised part was transferred to a Petri dish on a warming pad and kept moist. Bursal window was identified, then made an opening in this region large enough to invert the bursal sac without cutting into the oviduct running through. corpora lutea and follicles on each ovary were recorded. oviduct was isolated from bursa. The infusion needle is linked to the fallopian tube. A hemostatic forceps can be used to place the fallopian tube over the needle. Flush the fallopian tube with a syringe containing 5 ml, and oocytes were collected and observed at the count under microscope.

Evaluation of oocyte maturation of the collected oocytes was performed under stereomicroscope. Cumulus-Oocyte Complexes (COCs) were denuded with 0.1% hyaluronidase by gentle pipetting and they were observed without fixing and mounting and oocytes with nucleus and extruded first polar body (metaphase II) was counted and look under fluorescence microscope. The remaining oocytes denuded were fixed in 4% formaldehyde solution. For assessing in vitro maturation, oocytes were stained with 5μg/mL Hoechst 33342 for 10 min. The stained oocytes were mounted on a glass slide with a 100% of glycerol drops. Chromatin state and position as well as spindle formation of oocytes were evaluated under UV light to determine the stage of meiosis as follows (de Avila Rodrigues and Rodrigues, 2003): germinal vesicle (GV) stage: condensed chromatin, metaphase I (MI) stage: chromosomes were compact in a metaphase plate and migrating to the poles, metaphase II (MII) stage: the extrusion of the first polar body. The oocytes were classified as good, fair, poor, aged, or immature on the basis of both morphological evaluation and the stage of nuclear development.

The corpus luteum number, oocyte number, GV stage number, MI stage number, MII stage number and oocyte maturation rate of left and right ovaries were counted.

The estrus detection of bitch is shown in Figures 2-3. All dogs were in estrus after superovulation and had obvious estrus symptoms. Our results showed that 4-7 days after first intramuscular injection of PMSG, female dogs went from anestrus (Fig. 2A) to proestrus. In proestrus dogs appear restless, eating less and drinking more. The vulva turned redness and swelling with bloody discharge and palpation of hard vulva (Fig. 2B). The vaginal smears were characterized by a mixture of red blood cells, parabasal and intermediate cells and only a small number of superficial cells (<30%) (Fig. 3B), on the 10th day of the experiment, the follicles were clearly observed by ultrasonography (Fig. 3E). In estrus the bitch showed a strong desire to mate by showing a static reaction. The vulva becomes soft and vaginal secretions change from blood-red to colorless transparent or yellowish (Fig. 2C) and an increase in the percentage of superficial cells (70–90%) was detected in estrus (Fig. 3C). On the 18th day of the experiment, the corpus luteum formation in the ovary down to the ultrasonography (Fig. 3F) in the metestrus. The swelling of the female vulva gradually subsided and almost no secretion flowed out in metestrus (Fig. 2D).

Figure 4 shows the in vitro matured canine oocytes collected by flushing of fallopian tubes.

Table I. Evaluating the quality of ovulation on bitch after pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) superovulation treatment.

GV, germinalvesicle; MI, metaphase I; MII, metaphase II.

Evaluating quality of in vivo matured oocytes collected by flushing fallopian tubes after PMSG and hCG superovulation treatment

In this study, all bitches were in estrus state and showed ovulation. Number of corpus luteum, follicle and oocytes of 3 female dogs were counted in left and right ovary, respectively (Table I). All bitches produced corpus luteum, whereas only 2 bitches produced follicles. Moreover, all of the left and right ovaries of bitch produced oocytes (Fig. 5)

Evaluation of oocyte maturation of the collected oocytes from three bitch were performed under stereomicroscope (Table I). The mean luteal number and follicular number in the ovary were observed to be 21.7 and 7, respectively. An average of 21 oocytes were collected, with collection rate of 96.9%, including 4 at GV stage, 2.3 at MI stage and 14.7 at MII stage. The mean oocyte maturation rate was 69.8%.

The development of reproductive biotechnologies involving oocyte and embryo manipulation has advanced for many domestic species. These technologies include the in vitro production of embryos, embryo transfer, cloning, and transgenesis. However, the efficiency of IVM production in this species is very low, reproductive technologies for dogs generally require the use of in vivo-produced oocytes, at present. Although gonadotropins have been shown to be highly effective at inducing proestrus in bitches in some cases, in many other cases the ovulation rate was poor, pregnancy rates were not reported, and whelping rates were variable (Stornelli et al., 2012). Female dogs are monoestrus with an average estrous cycle of 6 months, and only 6 to 12 oocytes are produced in each cycle (Tsutsui, 1975; Lee et al., 2005; Reynaud et al., 2006). This in vivo production of mature oocytes is performed by collecting oocytes through flushing of the genital tract of a female after natural estrus or superovulation. Although the superovulation method is already well established for cattle (Bó and Mapletoft, 2014), pig (Angel et al., 2014), and sheep (Figueira et al., 2020), it is not yet completely developed for dogs, as this species is known to have a unique reproductive biology (Hirata et al., 2018).

The continuous low-dose (200 IU) PMSG intramuscular injection administered from days 1 to 8 and the high-dose (2000 IU) hCG intramuscular injection on day 12 induced superovulation. Our results showed that all dogs had marked estrus symptoms and ovulation, which is consistent with the results of Wright (1980). These estrus symptoms are consistent with the characteristics of normal estrus in dogs. Previous studies have shown that the estrus cycle is closely related to hormonal changes in the body. Follicle-stimulating hormone (FSH) is critical for ovarian folliculogenesis and essential for female fertility (Kumar, 2018). Although estradiol concentration were not measured based upon the hemotological profiles, it appears that the multiple-dose of 200IU PMSG was sufficient to induce follicular growth (Stornelli et al., 2012). In all bitches, the pre-ovulatory LH surge was accompanied by a pre-ovulatory FSH surge. We found that multiple-dose administration of pregnant mare serum gonadotropin in dogs facilitates follicular formation and we hypothesis that LH surge.

In dogs, ovulation is assumed to occur approximately 2 to 3 days after the preovulatory luteinizing hormone (LH) surge (Phemister et al., 1973; Concannon et al., 1977; Wildt et al., 1978), with ovulation being spread over 24 and 36 h (Concannon et al., 1977; Boyd et al., 1993). We injected the dogs with 2000 IU of hCG on day 11 to simulate the LH peak and ovulation. Corpora lutea were observed in the ovaries on day 18. Canine oviducts support the long-term survival of oocytes that undergo complete maturation, fertilization (in the oviductal isthmus), and development into the blastocyst stage. Therefore, canine oocytes may remain in the oviduct for 8.5 to 9 days (Holst and Phemister, 1971). Moreover, Lee et al. (2017) report that canine oviduct cell were beneficial to oocyte maturation. And that the use of hCG to induce ovulation in estrus induction protocols in bitches is quite controversial because they are spontaneous ovulators (Kutzler, 2010). In other spontaneously ovulating species (e.g., horses and cattle), hCG and GnRH are regularly used to time ovulation for appointed AI (Evans et al., 2006; Rensis et al., 2010). Therefore, the inclusion of hCG in estrus induction protocols in the bitch seems justified for timing ovulation rather than for induction of ovulation (Allen, 2008; Antonov and Georgiev, 2016). In our perliminary experiments, Oocytes collected surgically on day 22 are usually immature oocytes (Fig. 2A). Oocytes collected surgically on day 24 are usually aging oocytes (Fig. 2B). Thus, the oocytes were surgically harvested on day 23.

In this study, our aim was to collect mature oocytes by continuous injection of PMSG to superovulate the bitches. There is no direct correlation between oocyte maturation and ovulation timing. In our preliminary experiments, we identified the optimal time for obtaining mature oocytes during surgical procedures. Consequently, progesterone concentrations were not measured based on hematological profiles to determine the timing of ovulation.

The increase in oocyte number and maturation rate is beneficial to the development of the dog’s reproductive system. We hypothesized that long-term storage of oocytes in fallopian tubes is beneficial for oocyte maturation. We found that the mean luteal number and follicular number in the ovary were 21.7 and 7, respectively. An average of 21 oocytes was collected, and the oocyte collection rate was 96.9%, including 4 at the GV stage, 2.3 at MI, and 14.7 at MII. The mean oocyte maturation rate was 69.8%. Reynaud et al. (2005) reported that only 4% of ovulating dogs may be multi-oocyte, with two to five oocytes per follicle. this explains the lower number of corpora lutea than of oocytes in the dogs ovaries.

In summary, dogs can obtain a large number of high-quality mature oocytes by means of superovulation method; thus, this is an effective tool to understand the unique reproductive characteristics of dogs.

In this study, multiple repeat administrations of PMSG were, for the first time, used to induce superovulation in dogs. All dogs showed marked estrus and ovulation. The average oocyte number was 21. The mean oocyte maturation rate was 69.8%. This study provides basic data for future studies on the reproductive physiology of dogs.

This work was supported by the National Natural Science Foundation of China (No. 31860297).

This study was approved from Institutional Review Board of Yanbian University, Yanji, Jilin, China (IRB 20130310).

The animal experimental procedures of this study were approved by the Committee on the Ethics of Animal Experiments at Yanbian University (Approval ID: 20130310). All experiments were performed in accordance with relevant guidelines and regulations of ‘the instructive notions with respect to caring for laboratory animals’ issued by the Ministry of Science and Technology of the People’s Republic of China.

The authors have declared no conflict of interest.