Advances in complementary livestock artificial reproductive techniques
Keywords:
Assisted reproductive techniques, CRISPR-Cas9, embryo biopsy, estrus detection, reverse sex sorted semen, spermatogonial stem cell transplant
Abstract
Assisted reproductive techniques are beneficial to increasing animal agricultural production and improving veterinary medicine. Foundational techniques (e.g. artificial insemination and in vitro embryo production) have paved the way for both complementary and adjunct techniques to arise as technologies and research advance. Female/offspring focused technologies include those to increase precision of estrus detection, genomic selection through embryo biopsy, and gene editing via ‘clustered regularly interspaced short palindromic repeats’ (CRISPR) and CRISPR associated protein 9 (Cas9), whereas male focused technologies include sex sorting after freezing and thawing (‘reverse sex sorted semen’), multisire straws for artificial insemination, and spermatogonial stem cell transplantation. Although improvement in efficiency and feasibility is paramount, these recently developed and emerging techniques will likely become important to ensuring that animal products are produced efficiently and humanely. This review highlights these newer advances, with a particular focus on how they may impact mammalian agriculture and veterinary medicine.
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References
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11. Szenci O: Accuracy to predict the onset of calving in dairy farms by using different precision livestock farming devices. Animals 2022;12:2006. doi: 10.3390/ani12152006
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24. Qanbari S: On the extent of linkage disequilibrium in the genome of farm animals. Front Genet 2020;10:1304. doi: 10.3389/fgene.2019.01304
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26. Macedo GG, Mingoti RD, Batista EOS, et al: Profile of LH release in response to intramuscular treatment with kisspeptin in Bos indicus and Bos taurus prepubertal heifers. Theriogenology 2019;125:64–70. doi: 10.1016/j.theriogenology.2018.10.011
27. Pasquariello R, Bogliolo L, Di Filippo F, et al: Use of assisted reproductive technologies (arts) to shorten the generational interval in ruminants: current status and perspectives. Theriogenology 2024;225:16–32. doi: 10.1016/j.theriogenology.2024.05.026
28. Khatkar MS, Moser G, Hayes BJ, et al: Strategies and utility of imputed SNP genotypes for genomic analysis in dairy cattle. BMC Genomics 2012;13:1–12. doi: 10.1186/1471-2164-13-538
29. Aliloo H, Clark SA: The impact of reference composition and genome build on the accuracy of genotype imputation in australian Angus cattle. Anim Prod Sci 2021;61:1958–1964. doi: 10.1071/AN21098
30. Gunderson KL, Steemers FJ, Lee G, et al: A genome-wide scalable snp genotyping assay using microarray technology. Nat Genet 2005;37:549–554. doi: 10.1038/ng1547
31. Dehnavi E, Mahyari SA, Schenkel F, et al: The effect of using cow genomic information on accuracy and bias of genomic breeding values in a simulated Holstein dairy cattle population. J Dairy Sci 2018;101:5166–5176. doi: 10.3168/jds.2017-12999
32. Suárez-Mesa R, Ros-Freixedes R, Pena RN, et al: Impact of the leptin receptor gene on pig performance and quality traits. Sci Rep 2024;14:10652. doi: 10.1038/s41598-024-61509-1
33. Whitelaw B, Lillico S: Increasing livestock farming sustainability using genome editing technology. The Biochemist 2022;44:9–12. doi: 10.1042/bio_2022_114
34. Whitworth KM, Green JA, Redel BK, et al: Improvements in pig agriculture through gene editing. CABI Agric Biosci 2022;3:1–16. doi: 10.1186/s43170-022-00111-9
35. Hallerman EM, Bredlau JP, Camargo LSA, et al: Towards progressive regulatory approaches for agricultural applications of animal biotechnology. Transgenic Res 2022;31:167–199. doi: 10.1007/s11248-021-00294-3
36. Petersen B: Basics of genome editing technology and its application in livestock species. Reprod Domest Anim 2017;52:4–13. doi: 10.1111/rda.13012
37. Jenko J, Gorjanc G, Cleveland MA, et al: Potential of promotion of alleles by genome editing to improve quantitative traits in livestock breeding programs. Genet Sel Evol 2015;47:1–14. doi: 10.1186/s12711-015-0135-3
38. Nolen RS: FDA approves gene-editing tech creating PRRS-resistant pigs. AVMA - News. Schaumburg, IL; J Am Vet Med Assoc 2025. p. 716.
39. Grossman MR: Who will regulate genetically engineered animals in the United States? Eur Food Feed L Rev 2021;16:322.
40. Entis E: Aquadvantage salmon: a case study in transgenic food. Anim Biotechnol 1998;9:165–170. doi: 10.1080/10495399809525906
41. Richards M: FDA approves first-of-its-kind intentional genomic alteration in line of domestic pigs for both human food, potential therapeutic uses. US FDA: 2020. Available from: https://www.aasv.org/2020/12/fda-approves-first-of-its-kind-intentional-genomic-alteration-in-line-of-domestic-pigs-for-both-human-food-potential-therapeutic-uses/ [cited 29 December 2022].
42. Dolgin E: First GM pigs for allergies. Could xenotransplants be next? Nat Biotechnol 2021;39:397–401. doi: 10.1038/s41587-021-00885-9
43. Singh AK, Griffith BP, Goerlich CE, et al: The road to the first fda-approved genetically engineered pig heart transplantation into human. Xenotransplantation 2022;29:e12776. doi: 10.1111/xen.12776
44. Anonymous: FDA says GM pigs safe to eat. Nat Biotechnol 2025;43:839–839. doi: 10.1038/s41587-025-02716-7
45. Seidel Jr G: Economics of selecting for sex: the most important genetic trait. Theriogenology 2003;59:585–598. doi: 10.1016/S0093-691X(02)01242-6
46. Underwood S, Bathgate R, Maxwell W, et al: Birth of offspring after artificial insemination of heifers with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm. Anim Reprod Sci 2010;118:171–175. doi: 10.1016/j.anireprosci.2009.08.007
47. De Graaf S, Evans G, Maxwell W, et al: Birth of offspring of pre-determined sex after artificial insemination of frozen–thawed, sex-sorted and re-frozen–thawed ram spermatozoa. Theriogenology 2007;67:391–398. doi: 10.1016/j.theriogenology.2006.08.005
48. Fernandez-Novo A, Santos-Lopez S, Barrajon-Masa C, et al: Effect of extender, storage time and temperature on kinetic parameters (casa) on bull semen samples. Biology (Basel) 2021;10:806. doi: 10.3390/biology10080806
49. Li X-X, Wang M, Chen H-H, et al: Flow cytometric and near-infrared raman spectroscopic investigation of quality in stained, sorted, and frozen-thawed buffalo sperm. Anim Reprod Sci 2016;170:90–99. doi: 10.1016/j.anireprosci.2016.04.008
50. Woods K: Sexed semen could be the next drought mitigation strategy for beef. Australia; Beef Central. Jon Condon and James Nason: 2023. Available from: https://www.beefcentral.com/genetics/sexed-semen-could-be-the-next-drought-mitigation-strategy-for-beef/ [cited 24 November 2025].
51. Eaglen S: Why use pooled semen? Ag Proud. Jerone, ID; Progressive Publishing: 2023.
52. Baruselli PS, Ferreira R, Sá Filho MFd, et al: Using artificial insemination v. Natural service in beef herds. Animal 2018;12:s45–s52. doi: 10.1017/S175173111800054X
53. Zuidema D, Kerns K, Sutovsky P: An exploration of current and perspective semen analysis and sperm selection for livestock artificial insemination. Animals 2021;11:3563. doi: 10.3390/ani11123563
54. Cooke RF, Daigle CL, Moriel P, et al: Cattle adapted to tropical and subtropical environments: social, nutritional, and carcass quality considerations. J Anim Sci 2020;98:skaa014. doi: 10.1093/jas/skaa015
55. Teodoro R, Madalena F, Smith C: The value of f1 dairy Bos taurus- Bos indicus embryos for milk production in poor environments. J Anim Breed Genet 1996;113:471–482. doi: 10.1111/j.1439-0388.1996.tb00637.x
56. Oatley J: Refinement of surrogate sire breeding technology in cattle. Proc Plant and Animal Genome Conference/PAG 31. PAG, 12–17 January 2024.
57. Ciccarelli M, Giassetti MI, Miao D, et al: Donor-derived spermatogenesis following stem cell transplantation in sterile nanos2 knockout males. Proc Natl Acad Sci USA 2020;117:24195–24204. doi: 10.1073/pnas.2010102117
2. Ferré L, Kjelland M, Strøbech L, et al: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods. Animal 2020;14:991–1004. doi: 10.1017/S1751731119002775
3. Lamb GC, Mercadante VRG: Synchronization and artificial insemination strategies in beef cattle. Vet Clin North Am Food Anim Pract 2016;32:335–347. doi: 10.1016/j.cvfa.2016.01.006
4. Galli C, Duchi R, Crotti G, et al: Bovine embryo technologies. Theriogenology 2003;59:599–616. doi: 10.1016/S0093-691X(02)01243-8
5. Putney D, Drost M, Thatcher W: Influence of summer heat stress on pregnancy rates of lactating dairy cattle following embryo transfer or artificial insemination. Theriogenology 1989;31:765–778. doi: 10.1016/0093-691X(89)90022-8
6. Koziol JH, Armstrong CL: Manual for breeding soundness examination of bulls. Montgomery, AL; Society for Theriogenology: 2018.
7. Perry GA, Smith MF: Keys to successful estrus synchronization and artificial insemination programs. Proc Appl Reprod Strateg Beef Cattle. Virtual: 2020; p. 47–74.
8. Vishwanath R, Moreno J: Semen sexing–current state of the art with emphasis on bovine species. Animal 2018;12:s85–s96. doi: 10.1017/S1751731118000496
9. Matoba S, Zhang Y. Somatic cell nuclear transfer reprogramming: mechanisms and applications. Cell Stem Cell 2018;23:471–485. doi: 10.1016/j.stem.2018.06.018
10. Stygar AH, Gómez Y, Berteselli GV, et al: A systematic review on commercially available and validated sensor technologies for welfare assessment of dairy cattle. Front Vet Sci 2021;8:634338. doi: 10.3389/fvets.2021.634338
11. Szenci O: Accuracy to predict the onset of calving in dairy farms by using different precision livestock farming devices. Animals 2022;12:2006. doi: 10.3390/ani12152006
12. Arney DR, Kitwood SE, Phillips CJC: The increase in activity during oestrus in dairy cows. Appl Anim Behav Sci 1994;40:211–218. doi: 10.1016/0168-1591(94)90062-0
13. Pahl C, Hartung E, Mahlkow-Nerge K, et al: Feeding characteristics and rumination time of dairy cows around estrus. J Dairy Sci 2015;98:148–154. doi: 10.3168/jds.2014-8025
14. Reith S, Hoy S: Relationship between daily rumination time and estrus of dairy cows. J Dairy Sci 2012;95:6416–6420. doi: 10.3168/jds.2012-5316
15. Mills MD, Pollock AB, Batey IE, et al: Magnitude and persistence of higher estrus-associated temperatures in beef heifers and suckled cows. J Anim Sci 2024;102:skae079. doi: 10.1093/jas/skae079
16. Cooper-Prado MJ, Long NM, Wright EC, et al: Relationship of ruminal temperature with parturition and estrus of beef cows. J Anim Sci 2011;89:1020–1027. doi: 10.2527/jas.2010-3434
17. Chung H, Vu H, Kim Y, et al: Subcutaneous temperature monitoring through ear tag for heat stress detection in dairy cows. Biosys Eng 2023;235:202–214. doi: 10.1016/j.biosystemseng.2023.10.001
18. Miura R, Yoshioka K, Miyamoto T, et al: Estrous detection by monitoring ventral tail base surface temperature using a wearable wireless sensor in cattle. Anim Reprod Sci 2017;180:50–57. doi: 10.1016/j.anireprosci.2017.03.002
19. Riaz U, Idris M, Ahmed M, et al: Infrared thermography as a potential non-invasive tool for estrus detection in cattle and buffaloes. Animals 2023;13:1425. doi: 10.3390/ani13081425
20. Williamson N, Alawneh J, Bailey D, et al: Electronic heat detection. Proc South Island Dairy Event (SIDE) 2006; p. 1–9.
21. de Sousa RV, da Silva Cardoso CR, Butzke G, et al: Biopsy of bovine embryos produced in vivo and in vitro does not affect pregnancy rates. Theriogenology 2017;90:25–31. doi: 10.1016/j.theriogenology.2016.11.003
22. Mullaart E, Wells D: Embryo biopsies for genomic selection. In: Niemann H, Wrenzycki C: editors. Animal Biotechnology 2. Springer; Cham: 2018. p. 81–94. doi: 10.1007/978-3-319-92348-2_5
23. Shojaei Saadi HA, Vigneault C, Sargolzaei M, et al: Impact of whole-genome amplification on the reliability of pre-transfer cattle embryo breeding value estimates. BMC Genomics 2014;15:1–16. doi: 10.1186/1471-2164-15-889
24. Qanbari S: On the extent of linkage disequilibrium in the genome of farm animals. Front Genet 2020;10:1304. doi: 10.3389/fgene.2019.01304
25. Mueller ML, Van Eenennaam AL: Synergistic power of genomic selection, assisted reproductive technologies, and gene editing to drive genetic improvement of cattle. CABI Agric Biosci 2022;3:1–29. doi: 10.1186/s43170-022-00080-z
26. Macedo GG, Mingoti RD, Batista EOS, et al: Profile of LH release in response to intramuscular treatment with kisspeptin in Bos indicus and Bos taurus prepubertal heifers. Theriogenology 2019;125:64–70. doi: 10.1016/j.theriogenology.2018.10.011
27. Pasquariello R, Bogliolo L, Di Filippo F, et al: Use of assisted reproductive technologies (arts) to shorten the generational interval in ruminants: current status and perspectives. Theriogenology 2024;225:16–32. doi: 10.1016/j.theriogenology.2024.05.026
28. Khatkar MS, Moser G, Hayes BJ, et al: Strategies and utility of imputed SNP genotypes for genomic analysis in dairy cattle. BMC Genomics 2012;13:1–12. doi: 10.1186/1471-2164-13-538
29. Aliloo H, Clark SA: The impact of reference composition and genome build on the accuracy of genotype imputation in australian Angus cattle. Anim Prod Sci 2021;61:1958–1964. doi: 10.1071/AN21098
30. Gunderson KL, Steemers FJ, Lee G, et al: A genome-wide scalable snp genotyping assay using microarray technology. Nat Genet 2005;37:549–554. doi: 10.1038/ng1547
31. Dehnavi E, Mahyari SA, Schenkel F, et al: The effect of using cow genomic information on accuracy and bias of genomic breeding values in a simulated Holstein dairy cattle population. J Dairy Sci 2018;101:5166–5176. doi: 10.3168/jds.2017-12999
32. Suárez-Mesa R, Ros-Freixedes R, Pena RN, et al: Impact of the leptin receptor gene on pig performance and quality traits. Sci Rep 2024;14:10652. doi: 10.1038/s41598-024-61509-1
33. Whitelaw B, Lillico S: Increasing livestock farming sustainability using genome editing technology. The Biochemist 2022;44:9–12. doi: 10.1042/bio_2022_114
34. Whitworth KM, Green JA, Redel BK, et al: Improvements in pig agriculture through gene editing. CABI Agric Biosci 2022;3:1–16. doi: 10.1186/s43170-022-00111-9
35. Hallerman EM, Bredlau JP, Camargo LSA, et al: Towards progressive regulatory approaches for agricultural applications of animal biotechnology. Transgenic Res 2022;31:167–199. doi: 10.1007/s11248-021-00294-3
36. Petersen B: Basics of genome editing technology and its application in livestock species. Reprod Domest Anim 2017;52:4–13. doi: 10.1111/rda.13012
37. Jenko J, Gorjanc G, Cleveland MA, et al: Potential of promotion of alleles by genome editing to improve quantitative traits in livestock breeding programs. Genet Sel Evol 2015;47:1–14. doi: 10.1186/s12711-015-0135-3
38. Nolen RS: FDA approves gene-editing tech creating PRRS-resistant pigs. AVMA - News. Schaumburg, IL; J Am Vet Med Assoc 2025. p. 716.
39. Grossman MR: Who will regulate genetically engineered animals in the United States? Eur Food Feed L Rev 2021;16:322.
40. Entis E: Aquadvantage salmon: a case study in transgenic food. Anim Biotechnol 1998;9:165–170. doi: 10.1080/10495399809525906
41. Richards M: FDA approves first-of-its-kind intentional genomic alteration in line of domestic pigs for both human food, potential therapeutic uses. US FDA: 2020. Available from: https://www.aasv.org/2020/12/fda-approves-first-of-its-kind-intentional-genomic-alteration-in-line-of-domestic-pigs-for-both-human-food-potential-therapeutic-uses/ [cited 29 December 2022].
42. Dolgin E: First GM pigs for allergies. Could xenotransplants be next? Nat Biotechnol 2021;39:397–401. doi: 10.1038/s41587-021-00885-9
43. Singh AK, Griffith BP, Goerlich CE, et al: The road to the first fda-approved genetically engineered pig heart transplantation into human. Xenotransplantation 2022;29:e12776. doi: 10.1111/xen.12776
44. Anonymous: FDA says GM pigs safe to eat. Nat Biotechnol 2025;43:839–839. doi: 10.1038/s41587-025-02716-7
45. Seidel Jr G: Economics of selecting for sex: the most important genetic trait. Theriogenology 2003;59:585–598. doi: 10.1016/S0093-691X(02)01242-6
46. Underwood S, Bathgate R, Maxwell W, et al: Birth of offspring after artificial insemination of heifers with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm. Anim Reprod Sci 2010;118:171–175. doi: 10.1016/j.anireprosci.2009.08.007
47. De Graaf S, Evans G, Maxwell W, et al: Birth of offspring of pre-determined sex after artificial insemination of frozen–thawed, sex-sorted and re-frozen–thawed ram spermatozoa. Theriogenology 2007;67:391–398. doi: 10.1016/j.theriogenology.2006.08.005
48. Fernandez-Novo A, Santos-Lopez S, Barrajon-Masa C, et al: Effect of extender, storage time and temperature on kinetic parameters (casa) on bull semen samples. Biology (Basel) 2021;10:806. doi: 10.3390/biology10080806
49. Li X-X, Wang M, Chen H-H, et al: Flow cytometric and near-infrared raman spectroscopic investigation of quality in stained, sorted, and frozen-thawed buffalo sperm. Anim Reprod Sci 2016;170:90–99. doi: 10.1016/j.anireprosci.2016.04.008
50. Woods K: Sexed semen could be the next drought mitigation strategy for beef. Australia; Beef Central. Jon Condon and James Nason: 2023. Available from: https://www.beefcentral.com/genetics/sexed-semen-could-be-the-next-drought-mitigation-strategy-for-beef/ [cited 24 November 2025].
51. Eaglen S: Why use pooled semen? Ag Proud. Jerone, ID; Progressive Publishing: 2023.
52. Baruselli PS, Ferreira R, Sá Filho MFd, et al: Using artificial insemination v. Natural service in beef herds. Animal 2018;12:s45–s52. doi: 10.1017/S175173111800054X
53. Zuidema D, Kerns K, Sutovsky P: An exploration of current and perspective semen analysis and sperm selection for livestock artificial insemination. Animals 2021;11:3563. doi: 10.3390/ani11123563
54. Cooke RF, Daigle CL, Moriel P, et al: Cattle adapted to tropical and subtropical environments: social, nutritional, and carcass quality considerations. J Anim Sci 2020;98:skaa014. doi: 10.1093/jas/skaa015
55. Teodoro R, Madalena F, Smith C: The value of f1 dairy Bos taurus- Bos indicus embryos for milk production in poor environments. J Anim Breed Genet 1996;113:471–482. doi: 10.1111/j.1439-0388.1996.tb00637.x
56. Oatley J: Refinement of surrogate sire breeding technology in cattle. Proc Plant and Animal Genome Conference/PAG 31. PAG, 12–17 January 2024.
57. Ciccarelli M, Giassetti MI, Miao D, et al: Donor-derived spermatogenesis following stem cell transplantation in sterile nanos2 knockout males. Proc Natl Acad Sci USA 2020;117:24195–24204. doi: 10.1073/pnas.2010102117
Published
2025-12-01
How to Cite
Klabnik , J., & DuBois , J. (2025). Advances in complementary livestock artificial reproductive techniques. Clinical Theriogenology, 17. https://doi.org/10.58292/CT.v17.12959
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Review Reports
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