In vitro embryo production via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) in pure and crossbred Japanese Hokkaido native ponies

Abstract

This study evaluated the viability of in vitro embryo production using ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) as breeding techniques for pure and crossbred Hokkaido native ponies (n = 9). Oocytes were collected using transvaginal ultrasound-guided follicle aspiration. ICSI was performed on in vitro matured oocytes using frozen semen. Embryonic cultures were monitored using time-lapse cinematography. Blastocysts were cryopreserved and, after thawing, were transferred non-surgically into recipient mares. Over nine OPU sessions, the mean number of aspirated follicles was 23.9 (range, 13–49). The oocyte recovery and maturation rates were 35.3% (76/215) and 61.5% (40/65), respectively. The cleavage rate was 57.5% (23/40). Of cleaved embryos, 56.5% (13/23) were arrested at the 4-cell to 8-cell stage, and five developed into early-blastocyst. Three embryos were transferred, resulting in a successful pregnancy. In conclusion, OPU–ICSI is a viable assisted reproductive technology for enhancing the population of Japanese native horses.

In vitro embryo production is more challenging for horses than for cattle. The main obstacles are oocyte zona hardening during in vitro maturation [1], the inability of sperm to penetrate in vitro matured oocytes because of inadequate capacitation induction in vitro [2], and embryo culture [3, 4]. Intracytoplasmic sperm injection (ICSI) has allowed considerable progress in in vitro equine embryo production [5, 6]. Moreover, ICSI allows for the efficient use of scarce or poor-quality semen for embryo production [7].

Ovum pick-up (OPU) using transvaginal ultrasound-guided aspiration of immature follicles is the most common procedure for collecting oocytes for ICSI [8]. Moreover, oocytes recovered through OPU could be stored (‘held’) at 22°C for 24 h without losing viability [9]. In vitro equine embryos are smaller, with minimal blastocoel fluid content and no confluent embryonic capsule, enabling them to tolerate cryopreservation better than larger in vivo embryos [4]. Cryopreservation reduces the need to monitor a large number of fresh embryo recipients. OPU-ICSI can be performed at any stage of the reproductive cycle or season, allowing embryo production from subfertile or infertile mares and stallions with a 56% likelihood of pregnancy [10].

There are eight native horse breeds in Japan, with the Hokkaido native pony being the most numerous, accounting for approximately 65% of the native horses. Hokkaido native ponies are popular for riding, are known for their calm temperament, and can survive in severely cold weather. However, the native horse population in Japan has declined over the past few decades. The OPU-ICSI breeding technique could effectively increase the production of native Japanese horses by utilizing recipient mares of other breeds. This study aimed to evaluate the potential of OPU-ICSI to produce in vitro embryos from pure and crossbred Japanese Hokkaido native pony mares.

The OPU data for pure and crossbred Japanese Hokkaido native ponies are listed in Table 1. Overall, 76 oocytes were recovered from 215 aspirated follicles during nine OPU sessions. The size of 156 aspirated follicles was less than 10 mm, 56 were 10–20 mm, and three were 20–30 mm. The average follicle aspiration per OPU was 23.9 (range, 13–49). At recovery, eight oocytes were denuded, two were degenerated, and one lacked cytoplasm; these 11 oocytes were not considered for in vitro maturation. Oocyte recovery and in vitro maturation rates were 35.3% (76/215) and 61.5% (40/65), respectively. The developmental stages of the ICSI embryos cultured in vitro are presented in Table 2. For the 40 oocytes on which ICSI was performed, the cleavage rate was 57.5% (23/40); one of these early embryos arrested at the 2-cell, five arrested at the 4-cell, eight arrested at the 8-cell, and four arrested at the ≥ 16-cell stage. However, five injected oocytes developed into early blastocysts. The percentage of embryos arrested at the 4-cell and 8-cell stages was 56.5% (13/23) of all cleaved embryos.

Table 1. Results of OPU from Japanese Hokkaido native pony and its crossbreed (Hokkaido native pony-Connemara cross, and Hokkaido native pony-Haflinger cross).

Breed	OPU session	Aspirated follicle	Average follicle aspiration (range)	Recovered oocyte	Recovery rate (%)	Maturation rate (%)
Hokkaido native pony	4	129	32.3 (13–49)	46	35.7 (46/129)	55.6 (20/36)
Crossbreed	5	86	17.2 (13–24)	30	34.9 (30/86)	69.0 (20/29)
Total	9	215	23.9 (13–49)	76	35.3 (76/215)	61.5 (40/65)

OPU, ovum pick-up.

Table 2. Developmental stages of the OPU-ICSI-derived Hokkaido native pony and its crossbreed (Hokkaido native pony-Connemara cross and Hokkaido native pony-Haflinger cross) embryos cultured in a non-humidifying incubator with time-lapse cinematography. A charge-coupled device (CCD) camera was set up to acquire images every 15 min.

Breed	ICSI number	Cleavage rate (%)	Arrested at				Development of blastocyst
			2-cell	4-cell	8-cell	≥ 16 cell
Hokkaido native pony	20	55.0 (11/20)	-	4	4	1	2
Crossbreed	20	60.0 (12/20)	1	1	4	3	3
Total	40	57.5 (23/40)	1	5	8	4	5

OPU, ovum pick-up; ICSI, intracytoplasmic sperm injection.

Of the five early blastocyst stage cryopreserved embryos, we failed to transfer one into the uterus of the recipient mare because of inappropriate loading into the transfer catheter, and another failed to survive the recovery culture following thawing. Pregnancy was confirmed in one of three transferred embryos at 13 days post-ovulation in the recipient. The oocyte origin of the embryo of successful pregnancy was from a pure Hokkaido native pony. Figure 1 shows a photomicrograph of a day 7 cryopreserved early blastocyst after thawing (Fig. 1A) and an ultrasonographic examination of the vesicle in the recipient mare. An 11.4 mm embryonic vesicle (Fig. 1B) was observed on day 13 of pregnancy, and an embryonic heart (Fig. 1C) was visible on day 28. Normal fetal development was observed until the preparation of this article on day 206 of gestation. The observed fetal heart rate and maternal plasma progesterone concentration were 114 beats per minute (bpm) and 3.0 ng/ml, respectively. Moreover, noticeable fetal movement was observed during transrectal ultrasonography.

Fig. 1.

Photomicrograph of an early-blastocyst immediately before transfer and transrectal ultrasonographic image of the vesicle in the pregnant recipient mare. The oocyte origin of this embryo was from a pure Hokkaido native pony. (A) A day 7 cryopreserved early-blastocyst after thawing; (B) Image of an embryonic vesicle (11.4 mm) in the uterus of a recipient mare at day 13 of pregnancy; (C) Embryonic heart rate measurement at day 28 of pregnancy. As of the writing of this article, fetal development at seven months of gestation was progressing normally.

In vitro equine embryo production via OPU-ICSI has advanced significantly in the last decade because of improvements in immature oocyte collection and maturation, embryo culture from zygote to blastocyst, cryopreservation, pregnancy, and foaling rates [4, 8,9,10]. For the first time, we applied the OPU-ICSI breeding technique to pure and crossbred Japanese Hokkaido native ponies. We produced five viable blastocysts and achieved a successful pregnancy. The oocyte origin of the embryo that developed successful pregnancy was from a pure Hokkaido native pony.

A non-humidified incubator with time-lapse cinematography was used in the present study to monitor preimplantation embryonic development during culture. Only a few studies [11,12,13,14] have monitored the time-lapse imaging of equine embryos derived via ICSI. It has been suggested that time-lapse image analysis of early mitotic timing combined with morphological assessment can be a non-invasive method to effectively evaluate equine embryo developmental competence to the blastocyst stage [11, 12, 14]. In the present study, the cleavage rate of injected oocytes was 57.5%, similar to a previous study performed in draft and non-draft breeds [11]. The percentage of embryos arrested at the 4-cell and 8-cell stages was 56.5% (13/23) of the total cleaved embryos in the present study, indicating that development was arrested at this stage for most embryos. In this respect, a recent study [15] showed the upregulation of embryonic genes at the 4-cell and 8-cell stages in equine in-vitro-produced embryos, indicating the onset of equine embryonic genome activation. Embryonic genome activation is a critical event in embryo development, and many embryos fail to progress beyond this stage because of the failure of genome activation.

The average number of follicles aspirated per OPU in the present study was 23.9, with oocyte recovery and maturation rates of 35.5% and 61.5%, respectively. In the present study, oocyte maturation and cleavage rates in pure and crossbred Japanese Hokkaido native ponies were comparable to those in Arabian and Warmblood mares [16]. The expected oocyte recovery rate from immature follicles by an established OPU team is between 50% and 70% [3, 8]. The lower oocyte recovery rate in the present study may be attributed to a lack of experience. We successfully established one pregnancy from three transferred embryos. When writing this article, fetal development at seven months of gestation had progressed normally. Transrectal ultrasonography revealed noticeable fetal movement with a heart rate of 114 bpm, indicating a normal pregnancy. We hope to achieve a healthy foal.

In conclusion, our first attempt at OPU–ICSI in pure and crossbred Japanese Hokkaido native ponies successfully produced viable embryos, resulting in a confirmed pregnancy. Further improvements, particularly in oocyte recovery and embryo culture, could enhance the effectiveness of OPU-ICSI as an assisted reproductive technique to increase the Japanese native horse population.

Methods

The study protocol was approved by the Animal Welfare and Ethics Committee of Obihiro University of Agriculture and Veterinary Medicine, Japan (24-154). Nine healthy mares (Hokkaido native ponies = 4, Hokkaido native pony-Connemara cross = 3, Hokkaido native pony-Haflinger cross = 2) aged 3–12 years were used. They were kept in a pasture attached to a roofed shelter at the Horse Research Farm, Obihiro University of Agriculture and Veterinary Medicine, and fed hay, compressed hay (Hokkaido Horse Feed, Hokkaido, Japan), and balanced pellets (Stamm 30; Hallway Feeds, Lexington, KY, USA). For ICSI, frozen semen from Ireland-originating Connemara ponies was used. Connemara ponies have a good temperament and are well-known for being excellent riding horses. Hokkaido native ponies are also popular riders. Thus, the resulting crossbreed of the Hokkaido native pony and Connemara pony might produce an ideal riding breed in Japan.

Nine OPU sessions were conducted, one for each of the nine donor mares. A transrectal ultrasound examination of the ovaries was performed 1–3 days before the date of OPU to evaluate the number and size of follicles. After routine sedative, analgesic, and antibiotic pretreatment, an ultrasound probe (GE Healthcare Japan Corporation, Tokyo, Japan) with a 12G double-lumen needle (Minitube GmbH, Tiefenbach, Germany) was introduced into the vagina. Immature follicles were punctured, and fluid was aspirated using a vacuum pump (Peli Products S.L.U., Barcelona, Spain) connected to the lumen of the inner needle. Each follicle was flushed eight times with a follicle-size-dependent volume of commercial OPU recovery medium (EquiPlus, Minitube). The flushing fluid was transported to the ICSI laboratory within 10 min of completing OPU and filtered through an embryo filter (EZ Way Filter; SPI-MFG, Inc., Canton, TX, USA). The residual fluid was examined under a stereomicroscope (Nikon SMX1500; Nikon, Tokyo, Japan) to isolate oocytes.

For oocyte maturation, the cumulus-oocyte complexes (COCs) were transferred into 200 µl of Medium 199 (Earle’s salts, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 1% penicillin-streptomycin mixed solution (Nacalai Tesque), 5 mAU/ml FSH (Antrin R10; Kyoritsu Seiyaku, Tokyo, Japan), and 10% fetal bovine serum (Gibco), and incubated in 5% CO₂ at 37°C for 28–30 h. After maturation, the COCs were gently pipetted using a fine glass capillary to remove cumulus cells. A small amount of frozen-thawed semen from a Connemara pony stallion was transferred into a droplet (5 µl) of Toyoda–Yokoyama–Hosi (TYH) medium [17] under paraffin oil (Nacalai Tesque) in a petri dish for ICSI. However, in the present study, TYH medium was supplemented with 20 mM HEPES-Na (Nacalai Tesque), 5 mM NaHCO₃ (Nacalai Tesque), 0.1 mg/ml polyvinyl alcohol (Sigma-Aldrich Co., St. Louis, MO, USA), and 10% polyvinyl pyrrolidone (molecular weight: 360000, Nacalai Tesque) instead of bovine serum albumin. Morphologically normal and motile spermatozoa were aspirated into an injection pipette and immobilized using several piezo pulses (PMM-150FU; Prime Tech Ltd., Ibaraki, Japan). The immobilized spermatozoa were injected into MII oocytes.

The injected oocytes were washed thrice in KSOM supplemented with 3 mg/ml lipid-rich BSA (AlbuMAX; Gibco), 2% MEM Essential Amino Acid Solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 1% MEM Non-essential Amino Acid Solution (FUJIFILM Wako). Subsequently, groups of 2–5 injected oocytes were cultured in 20 µl of the same medium in a GPS dish (BIRR BioSciences b.v., Vreeland, Netherlands) for 7–10 days in a non-humidifying incubator with time-lapse cinematography (CCM-iBIS, ASTEC Co., Ltd., Fukuoka, Japan). A charge-coupled device (CCD) camera was set up to acquire images every 15 min.

ICSI-derived embryos were cryopreserved and thawed as previously described [18]. However, the blastocoel cavity was not punctured in the present study before cryopreservation. The recovery culture for the thawed embryos was continued for 6–7 h using the same culture medium and procedure described in the previous section for the culture of ICSI embryos. Embryos were then transferred non-surgically, as previously described [19], into the uterus of a recipient mare on day 4 after ovulation. Nine days after the transfer, pregnancy was diagnosed using transrectal ultrasonography, followed by weekly monitoring of conceptus development.

Conflict of interests

None of the authors have any financial or personal relationships that could influence or bias the content of this paper.