Biotechnologies of reproduction applied to dairy cattle production: Embryo transfer and IVF

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1 Biotechnologies of reproduction applied to dairy cattle production: Embryo transfer and IVF D. Bousquet 1, E. B. Burnside 2, and B. J. Van Doormaal 3 1 L Alliance Boviteq inc., Saint-Hyacinthe, Québec, Canada; 2 The Alliance Semex, Guelph, Ontario, Canada; 3 Canadian Dairy Network, Guelph, Ontario, Canada. Received 17 February 2003, accepted 30 July Can. J. Anim. Sci. Downloaded from by on 01/06/18 Bousquet, D., Burnside, E. B. and Van Doormaal, B. J Biotechnologies of reproduction applied to dairy cattle production: Embryo transfer and IVF. Can. J. Anim. Sci. 83: The objective of this paper is to review the utilization and outcome of reproductive biotechnologies in dairy cattle. Embryo transfer and IVF have their respective limits that influence their impact on a genetic improvement program. Embryo transfer is efficient and profitable in an artificial insemination breeding and selection program and IVF has great potential. Development of markers highly correlated with various economic traits will progressively be added to the present schemes to make them even more powerful. Key words: Reproductive technologies, embryo transfer, IVF, dairy cows, genetic Bousquet, D., Burnside, E. B. et Van Doormaal, B. J Biotechnologies de la reproduction appliquées à la production des bovins laitiers : transfert d embryon et FIV. Can. J. Anim. Sci. 83: Le but de cet article est de présenter les techniques et résultats de biotechnologies reliées à la reproduction. L investissement fait dans ces technologies doit être proportionnel au retour. En regard avec la situation actuelle, sur le plan technique, le transfert embryonnaire (TE) et la fécondation in vitro (FIV) ont leurs limites respectives qui influencent l impact qu ils peuvent avoir sur les programmes d amélioration génétique. Le transfert embryonnaire est efficace et profitable au niveau de la reproduction par insémination artificielle et des programmes de sélection. De plus, les FIV ont un très bon potentiel. Le développement de marqueurs correspondants aux différents traits économiques sera progressivement ajouté aux plans actuels pour les rendre encore plus puissants. Mots clés: Technologies de la reproduction, transfert d embryon, FIV, vaches laitières, génétique The reproductive biotechnologies discussed in the present paper are related to the embryo. They have been developed in parallel with our acquired knowledge regarding embryo development. Although not as widely used as artificial insemination, they have contributed substantially to the improvement of the dairy cattle industry through the production and reproduction of seedstock animals. Substantial commercial application of embryo transfer started in the early 1980s with the development of superovulatory regimens and efficient cryopreservation techniques. More recently (1990s), a new generation of embryo technology has developed, based on in vitro embryo production with the use of oocytes collected from live donors. The objectives of the present paper are to discuss the benefits that those embryo technologies have already given to the dairy industry and their potential for the future. EMBRYO TRANSFER (ET) AND IVF AROUND THE WORLD The International Embryo Transfer Society (IETS) has retrieved data on ET over the past 10 yr and more recently data Presented at the session Améliorer les productions animales grâce aux technologies reproductives. Improving Animal Production with Reproductive Physiology during the CSAS/ADSA/ASAS 2002 Joint Meeting, Québec City, QC, July To whom correspondence should be addressed (daniel. bousquet@umontreal.ca). 403 on IVF activities. In 2000, throughout the world approximately bovine in vivo derived embryos were transferred (Thibier 2001). Since the early 1990s, annual growth in the number of ET is a little more than 1%. In 2000, > cows were flushed, with an average of 5.9 transferable embryos per collection. Approximately 53% of the embryos were transferred frozen and the others fresh. In Canada and United States, a total of bovine in vivo derived embryos were transferred, with 74 and 37%, respectively, produced from dairy cows. Although the utilization of ET is relatively low compared to the use of AI, more than 95% of dairy bulls in AI centres are produced through ET (The Semex Alliance, personal communication), clearly demonstrating the importance of this technology to the dairy industry. With the advent of ET, every high-ranking bull dam can contribute one or more sons for progeny testing. This was not the case prior to availability of ET technology. International data retrieval of the utilization of IVF has started only in recent years. In North America, 1741 IVF embryos were transferred in 2000, corresponding to 1% of the global total. The popularity of these embryos is much higher in Europe, Asia and South America, where abbatoir- Abbreviations: ET, embryo transfer; IVF, in vitro fertilization; MAS, Marker Assisted Selection; MOET, multiple ovulation and ET; OPU, Ovum Pick Up; PCR, polymerase chain reaction

2 404 CANADIAN JOURNAL OF ANIMAL SCIENCE derived oocytes are predominantly used for IVF, as opposed to North America where antemortem oocyte retrieval is done on selected cows (Thibier 2001). EMBRYO TRANSFER AND SELECTION INTENSITY New opportunities for selection programs accompanied the introduction of ET technology. Although approximately 20% of the females do not respond to the superovulatory treatment and the number of transferable embryos varies among donors and collections, this technology has helped to increase prolificacy of selected donor cows. From one calf per year, the average number has increased to >10 with ET. This technology decreases generation intervals; at puberty, the heifer can produce three calves. In contrast, with AI only this productivity is usually obtained only when a cow reaches 6 yr of age. Embryo sexing is conducted on biopsies collected from embryos before transfer or freezing. The method used is based on polymerase chain reaction (PCR) technology and is readily applied under field conditions (Herr and Reed 1991). Determination of the sex of the embryo can be effective in >95% of the biopsies (Table 1). The pregnancy rate obtained with biopsied embryos transferred fresh varies according to the quality of the embryo and the use of heifers or cows as recipients [Table 1 and Darrow (2002)]. This technique is effective in herds where replacement heifers need to be produced within a short interval. Other groups reported 90% success rate in embryo sexing, with a pregnancy rate ranging from 58 to 71% after transfer of fresh biopsied embryos (Shea 1999). Embryo freezing has had a substantial impact on the industry. Using glycerol as cryoprotectant required removal of the embryo from the straw and washing before transfer (Miyamoto and Ishibashi 1978). However, the introduction of more permeable cryoprotectants (e.g., ethylene glycol and propylene glycol) eliminated the need for pretransfer embryo washing, enabling direct transfer to recipients after thawing (Voelkel and Hu 1992). The implementation of direct transfer has greatly increased the utilization of ET. Data collected by the Canadian Embryo Transfer Association (CETA) from its members on the pregnancy rate obtained with embryos transferred directly to recipients in 2000 are shown in Table 2. The main reason for using ET is to produce several progeny from superior cows; this enhances the quality of replacement heifers and bulls and thus intensifies genetic selection and hastens genetic gain. Using ET, genetic progress for milk production increased 20 to 30% (Nicholas and Smith 1983; Colleau 1985; Meuwissen 1990; Nicholas 1996). In Canada, the number of registered calves issued from ET increased from 2.5 to 4% between 1985 and 2002 (Table 3). Data on a total of 3172 donor cows for which at least one ET recovery was done in 2001 were retrieved from Holstein Canada and analysed to determine current trends in donor cow selection (Van Doormaal 2002). Analysed from various perspectives, all results indicated primary emphasis on overall type, both genetically and phenotypically. In terms of relative selection intensity at the genetic level, the emphasis on conformation was approximately 2.5 times that placed on Table 1. Embryo sexing results (using PCR) on biopsies collected from in vivo and in vitro produced embryos z In vivo In vitro Number Males 429 (48.8%) 1696 (55.9%) Females 407 (46.3%) 1168 (38.5%) Unknown 43 (4.9%) 168 (5.6%) Transferred fresh Pregnancies (day 30) 186 (57%) 532 (52.9%) Pregnancies (day 60) 179/324 (55.2%) 487/994 (48.9%) z L Alliance Boviteq Inc., September 1999 December Table 2. Summary of embryo transfer activity in Canada for the year 2000 z Number Donors Total Dairy (74%) Embryos collected (6.48/donor) Embryos frozen (4.4/donor) Pregnancy rate Fresh embryos / (62.1%) Frozen embryos y / (57.6%) z Reuben Mapletoft for CETA, CETA s Newsletter, y Embryos frozen in ethylene glycol. Table 3. Percentage of registered Holstein calves in Canada resulting from embryo transfer technology between 1985 and 2000 z Year Proportion of registrations (%) z Holstein Canada, yield traits (Fig. 1). It is interesting to note that the emphasis placed on this criterion has not changed over the past 10 yr. In 1991, 75.2% of donor cows were classified as Excellent or Very Good and in 2001, this percentage was 79.4%. On the other hand, fewer donor cows classified as Good or lower were selected in 2001 (1.6%) compared to 1991 (2.4%) (Van Doormaal 1991, 2002). Of the classified donor cows for 2001, 15.5% were Excellent while another 63.9% were Very Good. These percentages are very much higher than the proportions in the general Canadian Holstein population (0.8 and 16.0%, respectively). As a group, however, the average production performance based on all donor cows for their most recent lactation is higher than breed average (Van Doormaal 2002), indicating that on the whole the donors are above average for production as well as for type, although some lower-producing cows are being flushed (Table 4). Therefore, ET technology is not being utilized to its full potential to enhance genetic improvement. THE APPLICATION OF EMBRYO TRANSFER IN NUCLEUS PROGRAMS The 1980s and early 1990s were characterized by extensive research and development activity and some industrial

3 BOUSQUET EMBRYO TRANSFER AND IVF 405 Can. J. Anim. Sci. Downloaded from by on 01/06/18 Fig. 1. Relative genetic selection intensity for Holstein donor cows in 2001 compared to the active cow population (from Van Doormaal 2002). activity in so-called Multiple Ovulation and ET (MOET) nucleus programs. Early simulations (Nicholas and Smith 1983) suggested rapid rates of improvement in closed nuclei that would serve as a source of full-brother teams of sires and of AI proven sires. Later theoretical research (Leitch et al. 1995) revealed inbreeding rates were accelerated in three closed nuclei with reduced genetic variance and a slower rate of improvement. Attempts at MOET full-sib selection (Lohuis et al. 1990, 1993) were not always in agreement with theory, due to failure to get large enough full-sib families in a short interval, thus reducing selection accuracy and genetic progress. Industrial nucleus dairy breeding schemes have been attempted with varying results, from the early success of the Holland Genetics Scheme to closure of the Genus Nucleus Scheme, and abandonment of a major Nucleus Scheme proposed by Alta Genetics (due to financial challenges). IN VITRO EMBRYO PRODUCTION (IVF) The production of embryos using IVF presents two advantages over the conventional approach. First, it prolongs reproductive life of cows that do not respond to superovulation treatment or produce only unfertilized ova. It is, therefore, an integral tool to keep selected cows within a breeding scheme part of a selection program. It was demonstrated that within 50 d, the number of pregnancies can be four times higher than by using the conventional approach (Bousquet et al. 1999), thereby providing a means of increasing the pressure of selection on bull-dams. Using the IVF technology appropriately, Leitch et al. (1995) found that the annual genetic progress was 10 to 30% superior to conventional ET, chiefly because IVF offers greater flexibility in sires for mating, thus reducing inbreeding. The implementation of IVF technology allows the use of several bulls with one donor, since the control of fertilisation is made in vitro. This can be realised in one session of fertilisation as opposed to conventional ET, which is limited to one bull per session. Table 4. Production data of Canadian Holstein cows selected as donors in 2001 compared to the whole population in 2000 z Breed average in 2000 Donors in 2001 Milk Fat Protein Milk Fat Protein Average 305-d (kg) z Adapted from Van Doormaal (2002). Table 5. In vitro embryo production results with oocytes collected from mature cows or prepubertal 6-mo-old heifers z Number (%) Mature donor cows Prepubertal heifers Donors Collection Oocytes Cleaved (81.9%) 256 (76%) Embryos (47.3%) 66 (20%) Transfers (pregnancies) Fresh 125 (52%) z L Alliance Boviteq inc., September 1999 January Woolliams (1989) clearly demonstrated the importance of changing the bull at each embryo production in the conventional method. Second, IVF can be applied during different stages of reproduction, including the prepubertal period and during the first 3 mo of pregnancy. With the utilization of prepubertal heifers, it is possible to decrease the age of the donor female use, thereby contributing to a decreased generation interval. Using ultrasound-guided transvaginal collections, oocytes for IVF can be collected in heifers at 6 mo of age. In a study performed at L Alliance Boviteq on 32, 6-mo-old Holstein heifer calves, an average of two transferable embryos were produced per session (Table 5). The reduced yield of transferable embryos compared to that obtained with mature cows can be explained by the competency of collected oocytes. Galli et al (2001) also found decreased developmental capacity of oocytes collected from 6-mo-old

4 406 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 6. Estimated average number of calves produced per donor cow per year after oocyte collection and IVF or using the conventional method z IVM-IVF-IVC Mature Prebubertal Conventional donor cows y heifers Donor cows Frequency of collections 4/60 d 4/60 d 1/60 d Collections/year Number of transferable embryos/year Pregnancy rate 53% 53% 60% Number of calves z Adapted from Bousquet et al. (1999). y Mature donor infertile cows. calves compared to mature cows. In their study, 15 prepubertal calves were used in 82 oocyte collection sessions. On average, one transferable embryo per session was obtained. Even if the developmental capacity of the oocytes collected from calves is reduced compared to the ones from mature donor cows (average of four transferable embryos), the advantage still exists (Revel et al. 1995; Khatir et al. 1998). New hormonal treatments and preparation of oocyte donor animals are promising approaches to increase oocyte competency. In a recent study, up to 80% of oocytes collected from 14- to 16-mo-old heifers produced blastocysts after IVF (Blondin et al. 2002). Data from L Alliance Boviteq and elsewhere give the estimated potential of this technology compared to the conventional approach (Table 6). Using Ovum Pick Up (OPU) four times each 60-d period over 1 yr, 3.5 more calves will be produced in the case of mature cows and two more calves in prepubertal heifers. However, one might conclude that OPU should not be performed on prepubertal calves; since the progeny will be born before the performance of the donor is known, the accuracy of selection could be reduced. However, if a 6-mo-old-old heifer produces IVF embryos at that age, her sons will reach puberty at the stage when she has completed the first half of her 2-yr-old lactation. The use of Marker Assisted Selection (MAS) has the potential (if the markers are highly correlated with the desired phenotype) to enhance the power of the present-day breeding strategy (Georges 1998). Many genetic markers linked with QTL (quantitative trait loci) affecting traits of economic importance in livestock, including milk production, conformation and health have been identified and mapped during the past decade (Ashwell et al. 1998a, b; Van Tassell et al. 2000). Aggrey et al. (1999) found that one polymorphism of the bovine growth hormone receptor (GHR) gene was associated with milk-related traits in Holsteins. More recently, Plante et al. (2001) reported markers on chromosomes 20 and 26 affecting yield of milk fat and protein in Canadian Holsteins. Burnside et al. (2002) have identified four markers explaining significant protein yield variation in a selective genotyping, daughter design, for application in a commercial progeny-testing program. There are several stages at which MAS can be applied to accelerate genetic progress, notably, to designate from which daughters of bull dams embryos will be selected. Once the calves are born, MAS can be applied along with the present selection approach to identify bulls that will be reared for entry into the progeny test. Heifer calves can also be selected using the same tools and placed in the juvenile reproduction program. At 6 mo of age, their embryos produced with a selected bull can be biopsied and MAS applied on them to keep only the ones that carry the desired genes. A recent paper described a procedure for multiple genotype analysis, including gender, kappa-casein, growth hormone and prolactin from a single cell derived from bovine preimplantation embryos using PCR (Chrenek et al. 2001). The workers obtained an efficiency of genotyping by standard PCR from 88 to 91%. However, the efficiency of this approach could be increased by using the PEP-PCR method of DNA amplification (Garcia 2002). CONCLUSION Embryo technologies provide new possibilities for genetic selection schemes. Some have already been implemented, with ET being used on a regular basis for producing young sires for progeny testing. The combined use of Ovum Pick Up, followed by in vitro embryo production and marker assisted selection, will undoubtedly be the next generation of technologies applied in selection programs. ACKNOWLEDGEMENTS The authors gratefully acknowledge the professional and technical staff of The Alliance Boviteq Inc. for their active participation in producing the results presented in this article. Aggrey, S. E., Yao, J., Sabour, M. P., Lin, C. Y., Zadworny, D., Hayes, J. F. and Kuhnlein, U Markers within the regulatory region of the Growth hormone receptor gene and their association with milk-related traits in Holsteins. J. Hered. 90: Ashwell, M. S., VanRaden, P. M., Da Y., Rexroad, Jr., C. E. and Miller, R. H. 1998a. Detection of putative loci affecting conformational type traits in an elite population of United States Holsteins using microsatellite markers. J. Dairy Sci. 81: Ashwell, M. S., Da, Y., Van Tassell, C. P., VanRaden, P. M., Miller, R. H. and Rexroad, C. E. 1998b. Detection of putative loci affecting milk production and composition, health, and type traits in a United States Holsteins population. J. Dairy Sci. 81: Blondin, P., Bousquet, D., Twagiramungu, H., Barnes, F. and Sirard, M. A Manipulation of follicular development to produce developmentally competent bovine oocytes. Biol. Reprod. 66: Bousquet, D., Twagiramungu, H., Morin, N., Brisson, C., Carbonneau, G. and Durocher, J In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach. Theriogenology 51: Burnside, E. B., Pan,Y., Jansen, G. B., Plante, Y., Vandervoort, G. 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5 Colleau, J.-J Efficacité génétique du transfert d embryons dans les noyaux de sélection chez les bovins laitiers. Génét. Sél. Evol. 17: Darrow, M. D Pregnancy rates of sexed female fresh and frozen bovine embryos: a retrospective analysis. Theriogenology 57: 531 (Abstr.). Galli, C., Crotti, G., Notari, C., Turini, P., Duchi, R. and Lazzari, G Embryo production by ovum pick up from live donors. Theriogenology 55: Garcia, J. F Practical considerations of embryo manipulation: preimplantation genetic typing. Theriogenology 56: Georges, M Perspectives for marker assisted selection in dairy cattle breeding. Proceedings of the European AI Vets 10th meeting, Belgium. pp Herr, C. M. and Reed, K. C Micromanipulation of bovine embryos for sex determination. Theriogenology 35: Khatir, H., Lonergan, P. and Mermillod, P Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation. Theriogenology 50: Leitch, H. W., Smith, C., Burnside, E. B. and Quinton, M Effects of female reproductive rate and mating design on genetic response and inbreeding in closed nucleus dairy herds. Anim. Sci. 60: Lohuis, M., Smith, C. and Burnside, E. B Embryo results in a dispersed nucleus MOET/AI testing herd. Proceed. 4th World Congr. Genet. Appl. Livest. 14: Lohuis, M., Smith, C. and Dekkers, J. C. M MOET results from a dispersed hybrid nucleus program in dairy cattle. Anim. Prod. 57: Meuwissen, T. H. E The use of increased female reproductive rates in dairy cattle breeding schemes. Anim. Prod. 52: Miyamoto, H. and Ishibashi, T The protective action of glycols against freezing damage of mouse and rat embryos. J. Reprod. Fert. 54: BOUSQUET EMBRYO TRANSFER AND IVF 407 Nicholas, F. W and Smith, C Increased rates of genetic change in dairy cattle by embryo transfer and splitting. Anim. Prod. 36: Nicholas, F. W Genetic improvement through reproductive technology. Anim. Reprod. Sci. 42: Plante, Y., Gibson, J. P., Nadesalingam, J., Mehrabani- Yeganeh, H., Lefebvre, S., Vandervoort, G. and Jansen, G. B Detection of quantitative trait loci affecting milk production traits on 10 chromosomes in Holstein cattle. J. Dairy Sci. 84: Revel, F., Mermillod, P., Peynot, N., Renard, J. P. and Heyman, Y Low developmental capacity of in vitro matured and fertilized oocytes from calves compared with that of cows. J. Reprod. Fertil. 103: Shea, B. F Determining the sex of bovine embryos using polymerase chain reaction results: a six-year retrospective study. Theriogenology 51: Thibier, M The animal embryo transfer industry in figures: A report from the IETS data retrieval committee. IETS Newsletter, December. pp Van Doormaal, B. J To flush or not to flush? That is the question. Holstein Journal September/October: Van Doormaal, B. J Study of embryo transfer Holstein donor cows in [Online] Available: [5 August 2003]. Van Tassell, C. P., Ashwell, M. S. and Sonstegard, T. S Detection of putative loci affecting milk, health and conformation traits in a US Holstein population using 105 microsatellite markers. J. Dairy Sci. 83: Voelkel, S. A. and Hu, Y. X Direct transfer of frozenthawed bovine embryos. Theriogenology 37: Woolliams J. A Modification to MOET nucleus breeding schemes to improve rates of genetic progress and decrease rates of inbreeding in dairy cattle. Anim. Prod. 49: 1 14.

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