The physiology of multifactorial problems limiting the establishment of pregnancy in dairy cattle
Alexander C. O. Evans A B and Siobhan W. Walsh A
+ Author Affiliations
- Author Affiliations
A School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland.
B Corresponding author. Email: alex.evans@ucd.ie
Reproduction, Fertility and Development 24(1) 233-237 https://doi.org/10.1071/RD11912
Published: 6 December 2011
Abstract
The failure of cows to successfully establish pregnancy after insemination is an important limiting factor for the efficiency of dairy production systems. The physiological reasons for this are many and pertain to the post partum and early pregnancy periods. Cows that suffer severe negative energy balance after parturition are prone to diseases (including uterine infection) that are, in part, explained by reduced function of the immune system, having negative consequences for subsequent fertility. In high-producing dairy cows, the duration and intensity of oestrus is low as a consequence of low circulating oestradiol concentrations, and after insemination, high embryo mortality is the single biggest factor reducing calving rates. Embryo mortality occurs as consequences of poor oocyte quality (probably caused by the adverse metabolic environment) and by poor maternal uterine environment (probably caused by carry-over effects of uterine infection and low circulating progesterone concentrations). Immediate improvements in the fertility of lactating cows on many farms can be achieved by applying existing knowledge, but longer-term sustained improvement will require additional knowledge in many areas including the physiology of the tissues that contribute to reproduction.
Additional keywords: embryo loss, fertility, negative energy balance, progesterone.
References
Beam, S. W., and Butler, W. R. (1999). Effects of energy balance on follicular development and first ovulation in postpartum dairy cows. J. Reprod. Fertil. Suppl. 54, 411–424.| 1:CAS:528:DyaK1MXnslSit7k%3D&md5=9f453ed1872c358caa3d25f7cb14ce24CAS |
Butler, W. R. (2003). Energy balance relationships with follicular development, ovulation and fertility in postpartum dairy cows. Livest. Prod. Sci. 83, 211–218.
| Energy balance relationships with follicular development, ovulation and fertility in postpartum dairy cows.Crossref | GoogleScholarGoogle Scholar |
Chebel, R. C., Santos, J. E. P., Reynolds, J. P., Cerri, R. L. A., Juchem, S. O., and Overton, M. (2004). Factors affecting conception rate after artificial insemination and pregnancy loss in lactating dairy cows. Anim. Reprod. Sci. 84, 239–255.
| Factors affecting conception rate after artificial insemination and pregnancy loss in lactating dairy cows.Crossref | GoogleScholarGoogle Scholar |
Committee on Bovine Reproductive Nomenclature (1972). Recommendations for standardizing bovine reproductive terms. Cornell Vet. 62, 216–237.
Crowe, M. A. (2008). Fertility in dairy cows – the conference in perspective. In ‘Fertility in Dairy Cows – Bridging the Gaps’. (Eds M. D. Roayl, R. F. Smith and N. C. Friggens.) pp. 175–179. (British Society for Animal Science: Penicuik, UK.)
Dillon, P., Berry, D. P., Evans, R. D., Buckley, F., and Horan, B. (2006). Consequences of genetic selection for increased milk production in European seasonal pasture based systems of milk production. Livest. Sci. 99, 141–158.
| Consequences of genetic selection for increased milk production in European seasonal pasture based systems of milk production.Crossref | GoogleScholarGoogle Scholar |
Diskin, M. G., and Morris, D. G. (2008). Embryonic and early foetal losses in cattle and other ruminants. Reprod. Domest. Anim. 43, 260–267.
| Embryonic and early foetal losses in cattle and other ruminants.Crossref | GoogleScholarGoogle Scholar |
Diskin, M. G., Mackey, D. R., Roche, J. F., and Sreenan, J. M. (2003). Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle. Anim. Reprod. Sci. 78, 345–370.
| Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXksF2gt74%3D&md5=3607bc040c36ef890743e30529b825dfCAS |
Diskin, M. G., Parr, M., and Morris, D. G. (2012). Embryo death in cattle – an up-date. Reprod. Fertil. Dev. 24, 244–251.
| Embryo death in cattle – an up-date.Crossref | GoogleScholarGoogle Scholar |
Dobson, H., Smith, R., Royal, M., Knight, C., and Sheldon, I. (2007a). The high-producing dairy cow and its reproductive performance. Reprod. Domest. Anim. 42, 17–23.
| The high-producing dairy cow and its reproductive performance.Crossref | GoogleScholarGoogle Scholar |
Dobson, H., Walker, S. L., Morris, M. J., Routly, J. E., and Smith, R. F. (2008). Why is it getting more difficult to successfully artificially inseminate dairy cows? Animal 2, 1104–1111.
| Why is it getting more difficult to successfully artificially inseminate dairy cows?Crossref | GoogleScholarGoogle Scholar |
Dochi, O., Kabeya, S., and Koyama, H. (2010). Factors affecting reproductive performance in high milk-producing Holstein cows. J. Reprod. Dev. 56, S61–S65.
| Factors affecting reproductive performance in high milk-producing Holstein cows.Crossref | GoogleScholarGoogle Scholar |
Fair, T. (2010). Mammalian oocyte development: checkpoints for competence. Reprod. Fertil. Dev. 22, 13–20.
| Mammalian oocyte development: checkpoints for competence.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXitlagurg%3D&md5=f14ed197ba008e22494421dd33e2fd99CAS |
Galon, N., Zeron, Y., and Ezra, E. (2010). Factors affecting fertility of dairy cows in Israel. J. Reprod. Dev. 56, S8–S14.
| Factors affecting fertility of dairy cows in Israel.Crossref | GoogleScholarGoogle Scholar |
Galvao, K. N., Frajblat, M., Butler, W. R., Brittin, S. B., Guard, C. L., and Gilbert, R. O. (2010). Effect of early postpartum ovulation on fertility in dairy cows. Reprod. Domest. Anim. 45, e207–e211.
| 1:STN:280:DC%2BC3MzosFCltg%3D%3D&md5=5337efc6194bd66e289d67b119374d89CAS |
Garnsworthy, P. C., Fouladi-Nashta, A. A., Mann, G. E., Sinclair, K. D., and Webb, R. (2009). Effect of dietary-induced changes in plasma insulin concentrations during the early post partum period on pregnancy rate in dairy cows. Reproduction 137, 759–768.
| Effect of dietary-induced changes in plasma insulin concentrations during the early post partum period on pregnancy rate in dairy cows.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXosl2nur4%3D&md5=1206ad1166e806a5a5ead77e83606d12CAS |
Gilbert, R. O. (2012). The effects of endometritis on the establishment of pregnancy in cattle. Reprod. Fertil. Dev. 24, 252–257.
| The effects of endometritis on the establishment of pregnancy in cattle.Crossref | GoogleScholarGoogle Scholar |
Givens, M. D., and Marley, M. S. (2008). Infectious causes of embryonic and fetal mortality. Theriogenology 70, 270–285.
| Infectious causes of embryonic and fetal mortality.Crossref | GoogleScholarGoogle Scholar |
Gross, J., van Dorland, H. A., Bruckmaier, R. M., and Schwarz, F. J. (2011). Performance and metabolic profile of dairy cows during a lactational and deliberately induced negative energy balance with subsequent realimentation. J. Dairy Sci. 94, 1820–1830.
| Performance and metabolic profile of dairy cows during a lactational and deliberately induced negative energy balance with subsequent realimentation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXnvFChtLw%3D&md5=8d4b9e40a57d9ec41c6f370a4a1124f0CAS |
Grummer, R. R. (2007). Strategies to improve fertility of high yielding dairy farms: management of the dry period. Theriogenology 68, S281–S288.
| Strategies to improve fertility of high yielding dairy farms: management of the dry period.Crossref | GoogleScholarGoogle Scholar |
Gutierrez, C. G., Gong, J. G., Bramley, T. A., and Webb, R. (1999). Effects of genetic selection for milk yield on metabolic hormones and follicular development in postpartum dairy cattle. J. Reprod. Fertil. Abst Ser 24, 32..
Ingvartsen, K. L., Dewhurst, R. J., and Friggens, N. C. (2003). On the relationship between lactational performance and health: is it yield or metabolic imbalance that cause production diseases in dairy cattle? A position paper. Livest. Prod. Sci. 83, 277–308.
| On the relationship between lactational performance and health: is it yield or metabolic imbalance that cause production diseases in dairy cattle? A position paper.Crossref | GoogleScholarGoogle Scholar |
LeBlanc, S. J. (2008). Postpartum uterine disease and dairy herd reproductive performance: a review. Vet. J. 176, 102–114.
| Postpartum uterine disease and dairy herd reproductive performance: a review.Crossref | GoogleScholarGoogle Scholar |
LeBlanc, S. (2010). Assessing the association of the level of milk production with reproductive performance in dairy cattle. J. Reprod. Dev. 56, S1–S7.
| Assessing the association of the level of milk production with reproductive performance in dairy cattle.Crossref | GoogleScholarGoogle Scholar |
Leroy, J. L. M. R., Opsomer, G., De Vliegher, S., Vanholder, T., Goossens, L., Geldhof, A., Bols, P. E. J., de Kruif, A., and Van Soom, A. (2005). Comparison of embryo quality in high-yielding dairy cows, in dairy heifers and in beef cows. Theriogenology 64, 2022–2036.
| Comparison of embryo quality in high-yielding dairy cows, in dairy heifers and in beef cows.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2MnitlejtQ%3D%3D&md5=7b3fd3830d479919ba4d263a157d9ae6CAS |
Leroy, J. L., Opsomer, G., Van Soom, A., Goovaerts, I. G. F., and Bols, P. E. (2008). Reduced fertility in high-yielding dairy cows: are the oocyte and embryo in danger? Part I The importance of negative energy balance and altered corpus luteum function to the reduction of oocyte and embryo quality in high-yielding dairy cows. Reprod. Domest. Anim. 43, 612–622.
| Reduced fertility in high-yielding dairy cows: are the oocyte and embryo in danger? Part I The importance of negative energy balance and altered corpus luteum function to the reduction of oocyte and embryo quality in high-yielding dairy cows.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1cnksVeqsg%3D%3D&md5=f2b82cadeff433bad456da8441cae648CAS |
Lonergan, P. (2010). Using basic approaches to address problems in dairy reproduction. In ‘Reproduction in Domestic Ruminants’. (Eds M. F. Smith, M. C. Lucy, J. L. Pate and T. J. Spencer) pp. 377–386. (Nottingham University Press: Nottingham.)
Lopez, H., Satter, L. D., and Wiltbank, M. C. (2004). Relationship between level of milk production and estrous behavior of lactating dairy cows. Anim. Reprod. Sci. 81, 209–223.
| Relationship between level of milk production and estrous behavior of lactating dairy cows.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhsleltrw%3D&md5=0ff565da9086f9844869dd659e4ab2f8CAS |
Lucy, M. C. (2008). Functional differences in the growth hormone and insulin-like growth factor axis in cattle and pigs: implications for post-partum nutrition and reproduction. Reprod. Domest. Anim. 43, 31–39.
| Functional differences in the growth hormone and insulin-like growth factor axis in cattle and pigs: implications for post-partum nutrition and reproduction.Crossref | GoogleScholarGoogle Scholar |
Macdonald, K. A., Verkerk, G. A., Thorrold, B. S., Pryce, J. E., Penno, J. W., McNaughton, L. R., Burton, L. J., Lancaster, J. A. S., Williamson, J. H., and Holmes, C. W. (2008). A comparison of three strains of Holstein-Friesian grazed on pasture and managed under different feed allowances. J. Dairy Sci. 91, 1693–1707.
| A comparison of three strains of Holstein-Friesian grazed on pasture and managed under different feed allowances.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXktVeltLw%3D&md5=7be9ab0b68f77f30213c367373ef2da8CAS |
MacMillan, K. L. (2010). Recent advances in the synchronization of estrus and ovulation in dairy cows. J. Reprod. Dev. 56, S42–S47.
| Recent advances in the synchronization of estrus and ovulation in dairy cows.Crossref | GoogleScholarGoogle Scholar |
More, S. J., McKenzie, K., O’Flaherty, J., Doherty, M. L., Cromie, A. R., and Magan, M. J. (2010). Setting priorities for non-regulatory animal health in Ireland: results from an expert Policy Delphi study and a farmer priority identification survey. Prev. Vet. Med. 95, 198–207.
| Setting priorities for non-regulatory animal health in Ireland: results from an expert Policy Delphi study and a farmer priority identification survey.Crossref | GoogleScholarGoogle Scholar |
Mulligan, F. J., and Doherty, M. L. (2008). Production diseases of the transition cow. Vet. J. 176, 3–9.
| Production diseases of the transition cow.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXjs1Srsbc%3D&md5=2fdabd8420c7cccfd102e7610a4fe342CAS |
Norman, H. D., Wright, J. R., Hubbard, S. M., Miller, R. H., and Hutchison, J. L. (2009). Reproductive status of Holstein and Jersey cows in the United States. J. Dairy Sci. 92, 3517–3528.
| Reproductive status of Holstein and Jersey cows in the United States.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXnslGju7s%3D&md5=c5791cd6122b1104dec5fa06c32cdb00CAS |
Opsomer, G., Coryn, M., Deluyker, H., and de Kruif, A. (1998). An analysis of ovarian dysfunction in high yielding dairy cows after calving based on progesterone profiles. Reprod. Domest. Anim. 33, 193–204.
| An analysis of ovarian dysfunction in high yielding dairy cows after calving based on progesterone profiles.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXksVaiurw%3D&md5=cc5608db848459488ec01bc53b1cd51bCAS |
Peters, A. R. (1996). Embryo mortality in the cow. Anim. Breed. Abst. 64, 587–598.
Pryce, J. E., Royal, M. D., Garnsworthy, P. C., and Mao, I. L. (2004). Fertility in the high-producing dairy cow. Livest. Prod. Sci. 86, 125–135.
| Fertility in the high-producing dairy cow.Crossref | GoogleScholarGoogle Scholar |
Pursley, J. R., and Martins, J. P. N. (2012). Impact of circulating concentrations of progesterone and antral age of the ovulatory follicle on fertility of high producing lactating dairy cows. Reprod. Fertil. Dev. 24, 267–271.
| Impact of circulating concentrations of progesterone and antral age of the ovulatory follicle on fertility of high producing lactating dairy cows.Crossref | GoogleScholarGoogle Scholar |
Rizos, D., Ward, F., Duffy, P., Boland, M. P., and Lonergan, P. (2002). Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol. Reprod. Dev. 61, 234–248.
| Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xlt1Giug%3D%3D&md5=16e10a964f1ea09aa433817d64549866CAS |
Rizos, D., Carter, F., Besenfelder, U., Havlicek, V., and Lonergan, P. (2010). Contribution of the female reproductive tract to low fertility in postpartum lactating dairy cows. J. Dairy Sci. 93, 1022–1029.
| Contribution of the female reproductive tract to low fertility in postpartum lactating dairy cows.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXitlOjurc%3D&md5=fd7815dcedad679eaaa1a56e43660c61CAS |
Roche, J. F. (2006). The effect of nutritional management of the dairy cow on reproductive efficiency. Anim. Reprod. Sci. 96, 282–296.
| The effect of nutritional management of the dairy cow on reproductive efficiency.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtFSrurbN&md5=7d065505c503cf3b05b6a29cb9a1f928CAS |
Roche, J. R., Friggens, N. C., Kay, J. K., Fisher, M. W., Stafford, K. J., and Berry, D. P. (2009). Invited review: body condition score and its association with dairy cow productivity, health, and welfare. J. Dairy Sci. 92, 5769–5801.
| Invited review: body condition score and its association with dairy cow productivity, health, and welfare.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsFWisbbN&md5=804d681773c7a29642567374a4f51609CAS |
Roelofs, J., Lopez-Gatius, F., Hunter, R. H. F., van Eerdenburg, F. J. C. M., and Hanzen, C. (2010). When is a cow in estrus? Clinical and practical aspects. Theriogenology 74, 327–344.
| When is a cow in estrus? Clinical and practical aspects.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3cnntVakug%3D%3D&md5=13e362ff7f7576e155fdce221474b97aCAS |
Roth, Z., Arav, A., Bor, A., Zeron, Y., Braw-Tal, R., and Wolfenson, D. (2001). Improvement of quality of oocytes collected in the autumn by enhanced removal of impaired follicles from previously heat-stressed cows. Reproduction 122, 737–744.
| Improvement of quality of oocytes collected in the autumn by enhanced removal of impaired follicles from previously heat-stressed cows.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXos1Ohu74%3D&md5=4a20dbfc6d1a8a438a276f8d6f761b28CAS |
Saacke, R. G., Dalton, J. C., Nadir, S., Nebel, R. L., and Bame, J. H. (2000). Relationship of seminal traits and insemination time to fertilization rate and embryo quality. Anim. Reprod. Sci. 60–61, 663–677.
| Relationship of seminal traits and insemination time to fertilization rate and embryo quality.Crossref | GoogleScholarGoogle Scholar |
Sangsritavong, S., Combs, D. K., Sartori, R., Armentano, L. E., and Wiltbank, M. C. (2002). High feed intake increases liver blood flow and metabolism of progesterone and estradiol-17beta in dairy cattle. J. Dairy Sci. 85, 2831–2842.
| High feed intake increases liver blood flow and metabolism of progesterone and estradiol-17beta in dairy cattle.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xptlagsbc%3D&md5=1fa596de87d3e613858fe2b320282bb9CAS |
Sartori, R., Bastos, M. R., and Wiltbank, M. C. (2010). Factors affecting fertilisation and early embryo quality in single- and superovulated dairy cattle. Reprod. Fertil. Dev. 22, 151–158.
| Factors affecting fertilisation and early embryo quality in single- and superovulated dairy cattle.Crossref | GoogleScholarGoogle Scholar |
Sheldon, I. M., Lewis, G. S., LeBlanc, S., and Gilbert, R. O. (2006). Defining postpartum uterine disease in cattle. Theriogenology 65, 1516–1530.
| Defining postpartum uterine disease in cattle.Crossref | GoogleScholarGoogle Scholar |
Sheldon, I. M., Cronin, J., Goetze, L., Donofrio, G., and Schuberth, H. J. (2009). Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle. Biol. Reprod. 81, 1025–1032.
| Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsV2lt7vP&md5=01a60c8026ef758549aa650f1c9b7a60CAS |
Silke, V., Diskin, M. G., Kenny, D. A., Boland, M. P., Dillon, P., Mee, J. F., and Sreenan, J. M. (2002). Extent, pattern and factors associated with late embryonic loss in dairy cows. Anim. Reprod. Sci. 71, 1–12.
| Extent, pattern and factors associated with late embryonic loss in dairy cows.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD383kvFSkuw%3D%3D&md5=40cba1debbc09ccb98087fd853e5f564CAS |
Snijders, S. E. M., Dillon, P., O’Callaghan, D., and Boland, M. P. (2000). Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocyte development in dairy cows. Theriogenology 53, 981–989.
| Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocyte development in dairy cows.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c7ptlamsg%3D%3D&md5=93eade3732f314d3cbc826be0a2868dcCAS |
Sordillo, L. M., and Aitken, S. L. (2009). Impact of oxidative stress on the health and immune function of dairy cattle. Vet. Immunol. Immunopathol. 128, 104–109.
| Impact of oxidative stress on the health and immune function of dairy cattle.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXisVOgtbw%3D&md5=dd478f0172c341b3fc0886f8633c625eCAS |
Spencer, T. E., Sandra, O., and Wolf, E. (2008). Genes involved in conceptus–endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches. Reproduction 135, 165–179.
| Genes involved in conceptus–endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXit1yrtL4%3D&md5=221ea9d1418b824202c32d4d377bf676CAS |
Sreenan, J. M., and Diskin, M. G. (1983). Early embryonic mortality in the cow: its relationship with progesterone concentration. Vet. Rec. 112, 517–521.
| Early embryonic mortality in the cow: its relationship with progesterone concentration.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL3sXkvValu7s%3D&md5=b4a40a82ff7bdcae134e55006870a2b7CAS |
Taverne, M. A., and van der Weijden, G. C. (2008). Parturition in domestic animals: targets for future research. Reprod. Domest. Anim. 43, 36–42.
| Parturition in domestic animals: targets for future research.Crossref | GoogleScholarGoogle Scholar |
Thatcher, W., Santos, J., Silvestre, F., Kim, I., and Staples, C. (2010). Perspective on physiological/endocrine and nutritional factors influencing fertility in post-partum dairy cows. Reprod. Domest. Anim. 45, 2–14.
| Perspective on physiological/endocrine and nutritional factors influencing fertility in post-partum dairy cows.Crossref | GoogleScholarGoogle Scholar |
Vasconcelos, J. L. M., Silcox, R. W., Lacerda, J. A., Pursley, J. R., and Wiltbank, M. C. (1997). Pregnancy rate, pregnancy loss and response to heat stress after AI at two different times from ovulation in dairy cows. Biol. Reprod. 57, Abstr. 230..
Walsh, S. W., Williams, E. J., and Evans, A. C. O. (2011). A review of the causes of poor fertility in high milk producing dairy cows. Anim. Reprod. Sci. 123, 127–138.
| A review of the causes of poor fertility in high milk producing dairy cows.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3M3gt1eguw%3D%3D&md5=36dd0f85ec0e1518da83c701fce337afCAS |
Wathes, D. C., Cheng, Z., Fenwick, M. A., Fitzpatrick, R., and Patton, J. (2011). Influence of energy balance on the somatotrophic axis and matrix metalloproteinase expression in the endometrium of the postpartum dairy cow. Reproduction 141, 269–281.
| Influence of energy balance on the somatotrophic axis and matrix metalloproteinase expression in the endometrium of the postpartum dairy cow.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXisVehsbw%3D&md5=0d65dbcaa0b6319208440f97ffcd6fc5CAS |
