Ejaculated semen was collected from Kieng Sat, MươngTe, Co BinhThuan boars (7 - 10 months old) according to SATREPS/Vietnam protocol (2020) [10] . When the boar is mounted on the dummy, sow the spiral end of the penis with hands (gloved or bare, must be cleaned, dried and warmed up). To allow the boar to thrust through a clenched hand several times before applying a pressure, a hand pressure was applied to the spiral part of the penis to imitate the estrous sow’s cervix, stimulation ejaculation. When the penis is locked in the hand and the boar feels relaxed, a four-phase ejaculation follows in a few seconds, taking 5 to 10 minutes to completion. The first phase, called the pre-sperm fraction, contains clear seminal fluid, some gel, and dead sperm cells and is heavily contaminated with bacteria. It should not be collected. The next phase is the rich fraction sperm, easily recognized by its creamy-white color, although only 30 - 40 ml in volume contains a high density of spermatozoa. The third fraction, which is grayish with a lower density of spermatozoa, accounts for 50 - 70 ml of the collection. The fourth phase or post-sperm fraction provides a large semen volume peculiar. Up to the volume of 100 - 200 ml clear seminal plasma that is free of spermatozoa and gel is secreted from the accessory glands.

Because spermatozoa are very sensitive to rapid temperature changes, require a warm and dry collecting flask to safeguard semen fertility. In order to collect semen from boars, we used glass tubes (Sterile, dark colored, 300 - 500 ml) and semen filter paper. After ejaculation, semen were divided into the tubes and kept at 32˚C in a thermos bottle and transferred to the lab, immediately.

The collection spermatozoa from epididymis method were described by Kikuchi et al. (1998) [11] . Testes with attached epididymis were obtained from MươngTe, Kieng Sat and Co BinhThuan boars. Immediately, after removal from the scrotums, the testes were placed into plastic bags with sterile isotonic saline solution at room temperature and transferred to the laboratory within 1 h. In the laboratory, the epididymides were dissected and separated from the testis. Each cauda epididymis was dissected free, rinsed with 0.9% saline and placed into a 100 mm Petri dish. Caudae epididymides were held with forceps, and multiple incisions were made in the tubuli with a bistoury. Then, the spermatozoa were extruded from the caudaepididymidis by air pressure from a syringe and collected using 30 ml collecting solution. Sperm were diluted with a medium during storage at 15˚C for 2 - 3 hours.

Collected semen and epidiymal sperm was frozen using the method described in the method of Kikuchi et al. (1998) [11] .

Before freezing, collected sperm were placed on prewarmed glass sildes at 37˚C, and observed under a stereo microscopy for subjective evaluation of motility and morphological and evaluated the motility and morphology under a stereo microscope. Semen from boars which have more than 80% of total motility and 80% of normal spermatozoa were used. A sperm having an oval shaped head, an intact midpiece and an uncoiled single tail will be considered as a normal and healthy sperm. Sperms with normal morphology are able to swim well and in a straight line. Then, sperm concentration in samples was determined with a Neubauer chamber, with a dilution of 10 µl of semen to 990 µl of water. Thereafter the collected semen or epidiymal sperm was diluted to a ratio of 1:1 with collection medium prewarmed at 32˚C. Collection medium including 330 mM D-Glucose, 12.8 mM Sodium citrate, EDTA 9.9 mM, 14.3 mM Na-Bicarbonate, Penicillin (1000 unit/ml), Streptomycin (1 mg/ml).

After dilution with the collection medium, the semen was placed at 15˚C for 2 hours and then centrifuged at 3000 r/minute for 10 minutes at 15˚C. Then the supernatant was removed, and the pellet was stepwise resuspended in NSF-I extender to approximately 15 - 25 ml depending on the size of the sperm pellet. After dilution with NSF-I, the sperm was placed at 5˚C for an additional 2 hours. Thereafter, all steps were performed at 5˚C. Motility was verified again, and the sperm was diluted to 1:1 with NSF-II extender to a final volume of 30 - 50 ml. Then sperm was loaded to 0.25 ml plastic straws (Minutube) within 10 minutes which were immediately layered in the vapor above liquid nitrogen and kept there for 10 minutes. Then the frozen straws were moved under liquid nitrogen and stored there until use. For the final testing of motility, a frozen sperm straws from each lot was thawed by directly moving it with tweezers in a water bath at 37˚C for 20 seconds. Subsequently, the straw was wiped dry, opened with scissors and its content was placed on a microscope slide heated to 37˚C and evaluated for the parameters previously described for the semen immediately after collections.

In brief, we obtained pig ovaries from 6 - 8 months old crossbred gilts (Landrace × Large White) at a local slaughterhouse and transferred to the laboratory within 5 h in saline at 35˚C - 37˚C. The ovaries were washed 3 times in Dulbecco’s Phosphate Buffer Saline (DPBS; Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 0.1 mg/ml streptomycin sulfate (Sigma-Aldrich) and 100 units/ml penicillin G potassium (Sigma-Aldrich) at 37˚C and processed as follows. Cumulus-oocyte complexes (COCs) were collected by aspiration (at least 1 mm in diameter) into a collection medium consisting of Medium 199 (with Hank’s salts; Sigma-Aldrich) supplemented with 5% fetal bovine serum (FBS, Gibco; Invitrogen Corp., Carlsbad, CA, USA), 20 mM HEPES (Dojindo Laboratories, Kumamoto, Japan), and antibiotics 100 units/ml penicillin G potassium (Sigma-Aldrich) and 0.1 mg/ml streptomycin sulfate (Sigma-Aldrich)] in 60 mm petri dishes (Falcon 351007, Thomas Scientific, NJ, USA). COCs were collected under a stereo microscope. Oocytes with no apparent signs of lysis, having evenly granulated cytoplasm and at least 3 intact layers of cumulus cells were used.

Oocytes were cultured in a maturaion (porcine oocyte medium, POM) [12] . The POM was supplemented with, 10 ng/ml epidermal growth factor (EGF, Sigma-Aldrich), 10 IU/ml eCG (Serotropin; ASKA Pharmaceutical Co., Ltd., Tokyo, Japan), and 10 IU/ml hCG (500 units; Puberogen, Novartis Animal Health, Tokyo, Japan) throughout the entire IVM to the report of Van Khanh et al. (2021) [13] . The IVM medium was supplemented with 1 m Mdibutyrylc AMP (dbcAMP; Sigma) for the first 22 h of IVM to synchronise oocyte maturation. IVM culture was performed in 4-well dishes (NuncMultiDishes, Thomas Scientific) in 500-µl droplets of IVM medium covered by paraffin oil (Paraffin Liquid; NacalaiTesque) for 22 h in a condition of 5% CO₂, 5% O₂, and 90% N₂ at 39˚C. The COCs were subsequently cultured in the maturation medium without dbcAMP for an additional 22 - 24 h under the same atmosphere. Thirty to 50 COCs were cultured in each well.

In vitro fertilization and in vitro embryo culture were performed according to the method of Kikuchi et al. (2002) [14] . The medium used for IVF was Pig-FM (Suzuki et al., 2002) [15] containing 90 mmol/L NaCl, 12 mmol/L KCl, 25 mmol/L NaHCO₃, 0.5 mmol/L NaH₂PO₄, 0.5 mmol/L MgSO₄, 10 mmol/L sodium lactate, 10 mmol/L HEPES, 8 mmol/L CaCl₂, 2 mmol/L sodium pyruvate, 5 mmol/L caffeine and 5 mg/ml BSA (fraction V; Sigma). Before IVF, the outer layers of cumulus cells were removed from COCs after a short treatment with 0.1% hyaluroniadase (w/v). Then, the oocytes were washed three times in IVF medium and transferred to 90 µl IVF drops (10 - 20 oocytes/IVF drop) covered by mineral oil (Sigma). Frozen-thawed spermatozoa was placed in 7 ml of sperm washing medium (M199-Sigma, pH adjusted to 7.8) and centrifuged for 2 min at 2000 rpm. Then the pellet was re-suspended with 50 µl sperm washing medium and the motility of sperm was assessed subjectively under a stereo microscope. Only sperm samples with at least 30% motility were used for IVF. Then, 100 µl of the sperm suspension was transferred to a 30 mm Petri dish, covered by mineral oil and incubated at 37˚C for 15 min (Kikuchi et al., 1998). Thereafter, the sperm was stepwise diluted with IVF medium in a 4-well plate to achieve a concentration of 1 × 10⁶/ml. Then, 10 µl of the sperm dilution was introduced into 90 µl IVF droplets containing oocytes to achieve the final concentration of 1 × 10⁶/ml. The IVF drops were the incubated at 38.5˚C under 5% CO₂, 5% O₂ in humidified air for 6 hours. At 6 h after IVF, the spermatozoa and cumulus cells were removed from the surface of the zonapellucida by gentle pipetting with a fine glass pipette. After that, presumptove zygotes were cultured in 500 µl drops of PZM3 medium in 4-well dishes at 38.5˚C, 5% CO₂ in humidified air.

The insemination process was performed according to the method of Worwod (2007) [16] . The sows should be as acim and relaxed as possible before, during and after the insemination process. Clean the vulva of the sow or gilt by a damp cloth or paper towel. The semen is pulled into the sow by the rod. The reds are desgned to “lock” into the cervix of the sow with counterlockwise threads on the tip or rods with a rounded foam tip. Before gently inserting the rod into the vulva lubricate the tip with semen or a little lubricating jelly, and angle the rod tip upward. The rod with counterlockwise inserted the cervix. The rod with a rounded foam tip is inserted like threaded rod, but don’t need to be rotated, gently push until the foam tip catch in the foilds of the cervix. When the sow “accepts” the semen, insert bottle removed the tip into the end of the rod and gentle pressure, then the semen will begin to flow into the sow. Remove the bottle from the rod when it is becoming empty and add some air into the bottle, then reattach it and gently force the last of the semen from the rod and rotate the rod clockwise and withdraw it. Check the sow for standing heat 12 hours and 24 hours after last insemination, the best is the sow stops standing within 12 hours of insemination, the sow still standing maybe we inseminated too soon. The sows were checked for pregnancy by trans-abdominal ultrasound examination 28 days after inseminated. Pregnant sows were confirmed pregnant on Day 60.

All data were expressed as mean ± SEM values and the significant difference was checked by the ANOVA. P < 0.05 was defined as the significance level.

The quality of epididymis and ejaculated semen were assessed based on the average of the collected semen volume, sperm motility, sperm concentration, viable sperm and abnormal sperm (Table 1). The average volume of epididymal sperm was lower than that of ejaculation (from 2.98 to 3.98 ml vs from 71.32 ml to 79.24 ml, respectively). The quality of epididymal and ejaculated sperm of MuongTe, Kieng Sat, and Co BinhThuan pigs after freezing and thawing were based on percentages of motile sperm, viable sperm and abnormal sperm (Table 2). In this study, there was no significant difference in the sperm motility, viable sperm and abnormal sperm rates between the epididymis and ejaculation group (Table 2, P > 0.05).

We compared the feasibility of embryo fertilized with frozen-thawed epididymal

Five replicates were performed. Data are presented as means ± SEM.

Five replicates were performed. Data are presented as means ± SEM.

and ejaculated sperm in the Co BinhThuan breed. Based on the percentages of oocytes cleavage, blastocyst formation, blastocyst hatching and the cell number of blastocysts as described above. The results are summarized in Table 3. The rates of oocytes cleaved, blastocyst formation, and hatching blastocyst of ejaculated groups were significantly higher than that of the epididymal groups (60.11% vs 56.02%, 17.23%vs 14.31%, 3.78%vs 2.34%, respectively, P < 0.05). However, there was no significant difference in the average cell number of blastocysts among the groups (50.18 vs 50.02, respectively, P > 0.05).

We compared fertility of frozen-thawed epididymal and ejaculated sperm in the Co BinhThuan breed based on the pregnancy rate at Day 60 after aritifical insemination. The results are summarized in Table 4. The rate of pregnancy of ejaculated group was similar to that of the epididymal group (60% vs 60%).

In this experiment, we used the frozen-thawed sperm that collected by ejaculation method of MuongTe, Kieng Sat and Co BinhThuan pigs to do IVF. The results are showed in Table 5.

Six replicates were performed. Percentage values are presented as mean ± SEM. Different superscripts (a, b) denote a significant difference in the same row (P < 0.05).

Six replicates were performed. Percentage values are presented as mean ± SEM. Different superscripts (a, b) denote a significant difference in the same row (P < 0.05).

There was no significant difference in cleavage rate, blastocyst formation rate and the average cell number of blastocysts among the groups (Table 5, P > 0.05). However, the hatching blastocyst rate was significantly different between the groups. The percentage of hatching blastocysts of Co BinhThuan group was higher than MuongTe group (3.78% versus 2.59%, P < 0.05, respectively) but no difference between Co BinhThuan and Kieng Sat groups (respectively, 3.78% vs 3.01%, P > 0.05) or MuongTe and Kieng Sat groups (respectively, 2.59% vs 3.01%, P > 0.05).