Due to global climate change, population growth, and dietary improvement, animal protein is expected to be insufficient around 2050 (Westhoek et al., 2011), and many developed countries are attempting to increase meat production through genetic manipulation in livestock (Telugu et al., 2017). In addition, increased demand for protein globally is driven by socio-economic changes such as urbanisation and recognition of the role of protein for healthy aging (Delgado, 2003; Popkin et al., 2012). Although other alternatives such as cultured meat and textured vegetable protein are currently being developed (Young and Skrivergaard, 2020; Baune et al., 2022), people still need animal protein. However, despite the advantage of increasing the amount of meat, livestock born by manipulating genes cannot be distributed as the meat source in some countries (McFadden and Lusk, 2016) including Korea (Ministry of Trade, Industry and Energy of Korea, 2010). Therefore, in Korea, it is still necessary to increase the amount of meat through traditional genetic improvement rather than genetic manipulation.

There are native Korean cattle called Hanwoo. The coat is brown and both sexes are horned. It was formerly used as a working animal, but is now raised mainly for meat and become a premium product. Despite its high price, Hanwoo beef is preferred in Korean cuisine since it is typically fresher and of higher quality than cheaper imports. Kim et al. (2001) mentioned, “Hanwoo is regarded as a premium beef because of its high palatability and desired chewiness”. Since Koreans consider Hanwoo beef a cultural icon and one of the top-quality beefs of the world, it is used in traditional foods, popular holiday dishes, or as a special-day gift (Hur et al., 2008).

As a result of the implementation of ovum pick-up technology, called OPU, the embryo transfer (ET) industry has seen significant advancement in the utilization of in vitro produced (IVP) embryos (Sirard, 2018). A growing commercial interest has been sparked by the integration of genomic technology, which allows for shorter reproductive intervals and more accurate selection. In turn, the OPU-ET program is actively conducting numerous studies aiming to evaluate the quality of oocytes obtained through OPU and enhance efficiency for a more effective implementation of this technology (Pontes et al., 2011; Santos et al., 2016). In recent years, advancements in OPU technology and genomic selection have led to an increase in the use of IVP embryos. Therefore, the use of OPU in Korea has significantly increased to meet the demand for IVP embryos (Viana, 2022). Although OPU and IVP embryo are widely used in Hanwoo cattle, there is an imbalance between the growing demand for ET and the insufficient supply of high-capacity embryos, similar to the global trend in IVP-ET industry (Ferré et al., 2020). Thus, the OPU-ET program must increase efficiency by considering not only quantitative but also qualitative aspects.

This study was conducted with the aim of increasing meat production by selecting a giant cow of 1,000 kg or more by the aid of OPU and IVP embryo technologies to generate profits for Korean Hanwoo beef farmers.

Experimental designs

After the OPU from 1,100 kg of giant cow we have, the calves were produced in Hanwoo surrogate mothers with more than three delivery experiences through IVP system including in vitro fertilization (IVF), culture and ET.

Among 23 calves (7 females and 16 males) produced, six male heads were selected, and three heads of them were chosen as candidate sires and the rest three heads were raised for performance test.

The semen of three candidate sires from the age of 22 months were collected and froze, and the calves were produced by artificial insemination (AI) using those semen to Hanwoo cows who delivered more than twice. The calf was raised to the age of 30 to 33 months and performed progeny test.

All animals received humane care and all experiments with live animals were performed by a licensed veterinarian in accordance with the Korean Guidelines of Livestock Industry Act (article no. 11 and clause no. 1). IVP and ET procedure here is based on our team’s general protocol (Kim et al., 2017). In addition, delivery was always performed with human-assistance, and caesarean section was not implemented.

Oocyte recovery and in vitro maturation (IVM)

Immature Cumulus-oocyte complexes (COCs) were retrieved by OPU. The modified OPU was performed as previously described (Pontes et al., 2011). Briefly, each visible follicle (≥ 2 mm in diameter) was aspirated using Ibex® EVOTM (E.I. Medical Imaging, Loveland, CO, USA), a disposable 18 gauge × 90 mm hypodermic needle (Jeil tech Co., Seoul, Korea) connected to a 50 mL conical tube (Corning Life Sciences, Lowell, MA, USA), and a vacuum pump (Gast Manufacturing, Benton Harbor, MI, USA) with a negative pressure of 10-12 mL of water/min. The maturation step was conducted with IVMD 101, which is serum-free maturation medium (Functional Peptides Research Institute, Higashine, Japan) according to the company’s instructions. Briefly, COCs that were enclosed by more than three layers of compact cumulus cells and an evenly granulated ooplasm were selected for IVM. Selected COCs were cultured in 4-well culture dishes (Nunc, Roskilde, Denmark) containing 500 μL of IVMD 101 under warm, gas-equilibrated mineral oil for 20-22 h at 38.5℃, 5% CO₂.

IVP of bovine fertilized embryos and ET

Fertilized embryos were produced with IVF 100, which is serum-free fertilization medium (Functional Peptides Research Institute, Higashine, Japan) according to the company’s instructions. The expanded COCs were washed twice in IVF 100 and placed into 45 μL drops of IVF 100 under mineral oil. Frozen semen straws from three Hanwoo bulls confirmed high in carcass weight for their progeny were thawed in a 37℃ water bath and transferred to a 15 mL centrifuge tube with 4 mL IVF 100, and centrifuged at 600 g for 10 min. The supernatant was removed, re-suspended in 4 mL IVF 100 and centrifuged at 600 g for 10 min. After removal of the supernatant, 5 μL of the sperm suspension (1 × 10⁷ cells/mL) was introduced to the IVP drop, resulting in a final sperm concentration of 1 × 10⁶ cells/mL. Incubations were carried out at 39℃ in 5% CO₂ for 6 h. Then, the embryos were washed 3 times with IVMD 101 and cultured in an IVMD 101 drop for 24 h. Then, denuded fertilized oocytes were transferred to 100 μL of IVD 101, which is serum-free development medium (Functional Peptides Research Institute, Higashine, Japan), for 7 to 8 days at 38.5℃ in a humidified gas environment of 5% CO₂, 5% O₂ and 90% N₂. The culture drops were covered in mineral oil and 10 to 15 embryos were placed in each drop. Suitable embryos were selected under a light microscope and transferred to the recipients (one embryo per recipient).

Semen collection and semen freezing procedure

Using an artificial vagina, semen was collected from 3 Hanwoo bulls produced by IVP embryos as described above. Having collected the semen, the volume, color, pH, and sperm motility were evaluated to ensure the selection of normal semen. Sperm motility was assessed based on computer-assisted sperm analysis (QwikCheckTM Sperm Analyzer, Medical Electronic Systems Ltd., Israel). Sperm showing greater than 80% motility were used for subsequent freezing. The collected semen was diluted with semen freezing media (Optixcell; IMV imaging, France) and the concentrations of sperm per 0.5-mL straws were adjusted to 25 million. Following dilution, the diluted semen was stored at 4℃ for 3 to 4 h, after which the pre-cooled semen was loaded into 0.5-mL straws with SFM-1 (Fujihira Industry Co., Ltd., Tokyo, Japan) and sealed with polyvinyl alcohol powder. The loaded straw were then maintained at a height of 3 cm above the surface of liquid nitrogen for 14 min, after which, the cooled straw was immersed in liquid nitrogen and cryopreserved in a liquid nitrogen tank. Sperm in frozen-thawed semen at a concentration of 25 million per 0.5-mL straw were used for AI.

Detection of estrus in Hanwoo cows and AI

Hanwoo cow, which was delivered more than twice, was checked for estrus by standing and mucus around vagina at 08:00 and 16:00 every day. The cows, which were confirmed for estrus, were subjected to artificial insemination after 16 h based on the estrus confirmation time. Prior to performing AI, frozen straws were thawed in water at 38℃ for 40 sec and adapted to an AI gun with a sheath covered by a cover sleeve. AI was performed by a well-trained technician. After 90 days post-AI, pregnancy was confirmed by rectal palpation and trans-rectal ultrasound.

Performance and progeny tests

Total six male heads were selected, and three heads of them were raised for performance test and the rest three heads used as candidate sires. Three bulls for the performance test were fed for 30 months after castration at the age of 7 months, and total body weight, carcass weight, back fat thickness, sirloin cross-sectional extent, intramuscular fat, and marbling score were determined. The rating follows the guidelines of the Hanwoo board.

The rest of three candidate sires raised over 22 months and collected their semen for progeny test. After AI using their semen, the only male calves were raised to the age of 30 to 33 months after castration at the age of 7 months and performed progeny test. In the progeny test, only carcass weight and marbling score were displayed here.

From OPU to calves

As shown in Table 1, OPU was performed 13 times to collect 439 follicles, of which 352 oocytes were recovered (80.2%). A total of 256 recovered oocytes were used for actual IVM, of which 239 (93.4%) were cleaved after IVF. When the day of IVF was set to day 0, a total of 118 (46.1%) Day 7 was obtained and on Day 8, the total number of blastocysts was 128 (50.0%). Among them, 87 (68.0%) blastocysts showed good quality that can be used for ET. Of which 47 were transferred to surrogates and 23 calves were finally born. Of those calves, 16 were male and 7 were female and average of their body weight was 42.8 kg. This is 1.6-1.8 times higher than national average (National Institute of Animal Science, 2024).

Table 1. Calves obtained from ovum pick-up (OPU) from a giant cow with the aid of in vitro production and embryo transfer technologies.

OPU trials	Recovered oocytes/aspirated follicles (%)	Cleaved zygotes/cultured oocytes (%)	Day 7 & Day 8 blastocysts (%)	Transferrable blastocysts (%)*	Embryos transferred	Calved born**(male vs. female)(%/transferred)
13	352/439 (80.2)	239/256 (93.4)	118 (46.1) 128 (50.0)	87 (68.0)	47	23 (16 vs. 7) (48.9)

*Good quality embryos/Day 8 embryos..

**Average body weight: 42.8 kg [National average: 24 kg (female) and 27 kg (male); National Institute of Animal Science (2024)]..

Performance test of calves born by OPU and IVP

Table 2 shows the results of performance tests on three of the six male calves selected from calves born by OPU and IVP from a giant cow. The results of the performance test were significantly better than national average in all items that are indicators of production efficiency and quality of beef cattle, such as total body weight, carcass weight, back fat thickness, sirloin cross-sectional extent, intramuscular fat index, and marbling score (= meat quality grade) (Table 2). In particular, the carcass weight (615 kg) was more than 140 kg higher than the national average (467 kg; KAPE, 2024), and the meat quality was also rated 1++, the highest in all cattle.

Table 2. Performance test after 30 months of raise of calves* obtained from ovum pick-up from a giant cow with the aid of in vitro production embryo transfer technologies.

Calf number	Total body weight (kg)	Carcass weight (kg)	Back fat thickness (mm)	Sirloin cross-sectional extent (cm2)	Intra-muscular fat index**	Marbling score
1	999	611	25	107	7	1++
2	1,003	614	16	111	8	1++
3	1,016	621	14	115	9	1++
Average	1,006	615.3	18.3	111	8	All 1++
National average***	697.6	467.0	14.7	109.3	7.1	Only 22.2% of ox rated as 1++

*Castration at the age of 7 months..

**7 (16-17%), 8 (17-19%), 9 (19% and over)..

***Castrated (total body weight 500 kg and over): Korea Institute for Animal Products Quality Evaluation (KAPE, 2024)..

Progeny test of calves born by AI using giant cow-derived candidate sires

As the top requirement of Korean beef farmers in the field is to greatly improve the weight of beef cattle, we first selected large cows for oocyte donors and focused on heavyweights by using semen from a bull with a good body type for heavyweights. As shown in Table 3, in progeny test, the average birth weight of calves born after artificial insemination of semen from giant cow-derived candidate sires was 41 kg, which is remarkably higher than that of national average. After raised to the age of 30 to 33 months, the average weight of carcass of the progeny of the giant cow-derived candidate sire was 562.7 kg and it is approximately 100 kg higher than the average carcass (467 kg; KAPE, 2024) of castrated Hanwoo bull. This indicates that giant cow-derived progeny can provide an enhanced meat yield, which may contribute to addressing the anticipated global protein shortage. Marbling score is also higher than national average of Hanwoo cattle. Fig. 1 shows one of the candidate sires in this study (candidate sire number 2 of Table 3).

Table 3. Progeny test of calves born after artificial insemination of semen from giant cow-derived candidate sires.

Candidate sire number	Number of progeny*	Average birth weight of the progeny (kg)	Average carcass weight of the progeny (kg)	Marbling score ranges (average)**
1	14	40	534	1-1++ (1.53+)
2	23	41	593	1-1++ (1.48+)
3	17	42	561	1-1++ (1.59+)
Average	n/a	41	562.7	1.53+ (66.7% of ox rated as 1++)
National average	n/a	24 (female)*** 27 (male)***	467.0****	Only 22.2% of ox rated as 1++****

*Castration at the age of 7 months..

**For convenience, the average of marbling scores was calculated by counting the number of +. For example, if there were one 1++ and one 1+, the average in this table is expressed as 1.5+..

***National Institute of Animal Science (2024)..

****Castrated (total body weight 500 kg and over): Korea Institute for Animal Products Quality Evaluation (KAPE, 2024)..

Figure 1.One of the candidate sire (number 2 in Table 3). This Hanwoo bull weighs 1,700 kg and is currently being collected semen for cryopreservation.

The results of this study demonstrate the effectiveness of selecting and utilizing giant cows for the improvement of Hanwoo cattle, particularly in terms of meat yield and quality. By employing advanced reproductive techniques such as OPU and IVP, this study successfully produced calves with carcass weights and marbling score superior than the national averages.

The successful application of OPU and IVP techniques on giant Hanwoo cow provides a robust model for enhancing both meat yield and quality, thereby offering a viable solution to increasing beef production. It also offers economic benefits to the company with such sires. By generating a consistent source of revenue and improving the genetic stock of Hanwoo cattle, the approach outlined in this study aligns with the growing demand for sustainable agricultural practices that enhance productivity without relying on genetic modifications. We continue to make quality Hanwoo with excellent meat using the bulls we selected above.

From an animal welfare perspective, it is noteworthy that this approach complies with humane standards and provides an alternative to genetic modification, which remains controversial and restricted in Korea (Ministry of Trade, Industry and Energy of Korea, 2010). By focusing on traditional genetic improvement methods, this research aligns with both regulatory frameworks and consumer preferences within the region. Future studies should focus on the long-term genetic stability and health implications of this approach to ensure continued improvements in the Hanwoo breed.

This study lays a crucial foundation for further developments in Korean cattle farming, particularly as it relates to sustainable and high-yield beef production.

The authors thank the beef cattle farmers for participating in this study.

Conceptualization, S.R. and Y-G.J.; project administration and resources, Y-G.J.; methodology and investigation, Y-G.J., S.J., H.S. and Y.J.; data curation and validation, N.A. and S.R.; writing-original draft preparation, N.A. and S.R.; Writing-review and editing, S.R. and Y-G.J.

None.

Not applicable.

Not applicable.

Not applicable.

Not applicable.

No potential conflict of interest relevant to this article was reported.