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Production of embryos in vitro and its-impact on livestock production
THERIOGENOLOGY
PRODUCTION OF EHBRYOS IN VITRO AND ITS IMPACT ON LIVESTOCK PRODUCTION I. Gordon & K. H. Lul Department of Animal Science h Production, University College Dublin, Lyons Estate, Newcastle, County Dublin, Ireland ABSTRACT In Ireland, an attempt has been made to scale-up a laboratory procedure, which had yielded promising results, to meet the needs of extensive commercial exploitation of the technology of in vitro cattle embryo production. This paper examines some of the problems which have to be dealt with in bringing the embryo production technology to the point at which it becomes fully viable commercially. Much of the information included in the review is derived from on-going research and development work in the University; data are also derived from the work of Ovamass Ltd., a in 1987 to develop company established in Ireland the technology of embryo production on a commercial basis. Key words: cattle, in vitro, oocytes, embryo production.
young in limited numbers In recent years, have been produced in sheep (11, cattle (2) and pigs (3) by (IVF) of techniques involving the in vitro fertilization ovarian oocytes recovered from slaughterhouse animals and matured in vitro. Of the farm animals, it is in cattle that interest lies in greatest getting the the emerging technology of in vitro embryo production developed to the of commercial relevance. point of being Although nonmethods for surgical recovery obtaining embryos from superovulated donor cattle have been available for some are costly. such methods The ovaries of years, slaughterhouse cattle, on the other hand, could provide an appropriately low cost supply of oocytes that could be and cultured in fertilized vitro to the matured, transferable stage of development. In Dublin, a production system has been devised to provide such a low cost supply of cattle embryos using slaughterhouse cattle ovaries as the raw materials (4,5,6) and this has resulted in attempts to commercialise the procedure and through embryo transfer to apply the new technology on the farm. 1. Present Address: Ovamass Ltd.,Fethard, County Tipperary. Acknowledgements: The support of Ovamass Ltd is gratefully acknowledged.
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However, it is now evident that further developments and are required in bringing refinements embryo production technology to the point at which it can have significant commercial impact. Although oocytes in large numbers can be recovered from slaughterhouse ovaries, there are problems yet to be overcome in consistently producing transferable/ freezable quality embryos in quantity. For thought of any extensive application of non-surgical embryo transfer on the farm, there are several considerations relating to such techniques and the most appropriate transfer ways of husbandry problems dealing with the that arise, may particularly when the application is directed towards the induction of twin-calvings. PRODUCING EMBRYOS IN THE_LABORATOm Collection ofOvaries.‘----‘--The raw rnaterialszsoyed in the large-scale production of cattle embryos have been the ovaries of slaughterhouse heifer cattle. The original protocol involved bringing the ovaries from the abattoir in medium held at 30-37°C (1) but is now clear that the ovaries can be held at 200C for at oocytes are recovered without least 8 h before this compromising the acceptability of oocytes for maturation and fertilization (7). This permits much greater freedom in arranging production line work. Each bovine ovary contains many thousands of oocytes, but with current technology only a relatively small proportion follicle population is utilized. of the total vesicular dissection was initially employed in Although follicle reccvering oocytes from follicles (2-6 mm diameter), for routine use, aspiration is now the method of choice. The obvious advantage of aspiration iS in terms of speed of important in an embryo operation, which is especially production line system. Data in Table 1 show that the outcome at 48 h post-insemination, was not compromised by using aspiration rather than follicle dissection (8). COMPARISON OF FOLLICLE DISSECTION AND ASPIRATION TABLE 1. 4S METHODS OP OOCYTB RBC?~~_EMPLO_YED_IN.C_~_TTLE_IVF~,_ +--.--. Me t h o d Dissection Aspiration
Ovaries 892 646
Oocytes Recovered 5822 4511
Oocytes Cultured 3642 3310
Put to IVF
Cleavage Rate'
2974 2494
83.5% 82.4%
The principle of maintaining an intact cumulus-oocytecomplex (C-O-C) is regarded as an important one to observe at all times and evidence is available that this can have a genuine effect on the yield of embryos (9).
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THERIOGENOLOGY
The maximum number of usable oocytes recoverable per ovary, using follicles of 2 mm diameter and above, would be about 10 (8). Reports in the literature generally show some 40-50 vesicular follicles to be in the 2mm and above category but only about 50% of these would be capable of yielding an acceptable There is need oocyte. for much further information on the incidence and extent of follicular atresia in relation to follicle size and the reproductive status of the cattle that are being used as the source of ovaries. Cow ovaries have been found to yield a smaller number of acceptable oocytes than beef heifer cattle that are slaughtered at 2-3 years (8). There is also the question of the meiotic competence of oocytes in follicles that are one mm diameter and less in diameter. The data of Notlik 61 Fulka (10) indicate that bovine follicles up to 1.6 mm diameter may contain oocytes that are still growing and have not yet attained the state of being meiotically competent.Studies conducted in Ovamass by Tan & Lu (11) have shown that oocytes recovered from give follicles below 2mm diameter significantly lower yields of embryos after in vitro maturation and IVF. There is a need to examine procedures whereby growing oocytes obtained from the small follicles can be cultured to the point at which they do attain meiotic competence. In terms of the total vesicular follicle population in the bovine numbers present there are large the ovary, (12,13); challenge is one of devising appropriate ways of fully utilizing the oocytes which the contain. OOCYTE MATURATION.. In sheep. the requirement for follicle cell support in the Cf the oocyte was cytoplasmic maturation clearly demonstrated (14) and this same principle was applied in the initial Dublin work, using additional cumulus cells and hormone supplementation (15). This protocol was modified to exclude hormones on the basis of data from subsequent studies (16,17). In using additional cumulus cells in the current production system, there is no evidence of any temporary inhibition of meiosis in the bovine oocytes, such as that recorded by some workers (18) when cumulus cells have been added to culture medium prior to commitment of the oocytes to germinal vesicle break-down. A non-static routinely employed as a means of system is culture preventing the plating of cumulus cells and incubations are carried out at 39'C, the core temperature of the cow. (H-199) is currently supplemented The maturation medium with 20% estrous cow serum (ECS),this form of serum being employed rather than fetal calf serum (FCS) on the basis of earlier work (16).Subsequent results have not always shown similar evidence of an advantage(E).Elsewhere,studies have shown ECS to have a favourable effect on IVF in comparison with sera obtained at ovulation and 24 h after ovulation
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(19) i others have reported pro-estrous serum to have the most favourable effect on IVF in their studies.There are obviously steroids,peptide hormones and growth factors in bovine serum whose role in oocyte maturation remains to be determined. Medium 199 is generally employed in maturing bovine oocytes and is usually supplemented with gonadotropins (FSH/LH) and estradiol; in sheep, such supplementation had proved to be essential in cattle some work has shown a (14) and favourable effect (20,21).In Dublin, although oocytes are routinely matured in the absence of hormones, there is some effect of estradiol evidence of a favourable in the maturation medium which becomes evident at the 7-day stage of development (22). There is clearly a need for much further work in examining the hormonal requirements of oocyte maturation in the cow, since present evidence is somewhat conflicting. In Dublin, immature eggs are cultured for 24-26 h in groups of 40 in 2 ml volumes of maturation medium. The routine maturation procedure has been shown to be capable of yielding more than 90% of oocytes at metaphase II (17). SPERM CAPACITATIOW, For an effective IVF system,it is clearly essential to have a reliable means of preparing sperm. In Dublin, although several procedures for capacitating bull sperm have been examined, the most effective to date is the method based on that reported by Parrish et al. (23) who demonstrated that heparin could increase IVF frequencies: details of the heparin treatment as used in Dublin have been reported elsewhere used in (15,24). The semen routine embryo production is obtained from local cattle breeding stations and semen straws from several bulls within the selected breed are pooled to provide sperm for the IVF. A 60-minute swim-up procedure is used to isolate a highly motile sperm population prior to washing and a 15 min. incubation period with heparin. In terms of factors affecting the outcome of of difficulty in IVF, bull variability has been one source the Dublin work, with certain individual bulls or even bulls groups of within a breed showing much higher fertilization frequencies than others (25). IN VITRO FERTILIZATThe fertilization of matured oocytes has been routinely carried out in microdroplets of HEPES-TAPES medium droplets contain 10 normally oocytes but (pH,7.S); considerably larger numbers (UP to 50) can be included without compromising the outcome of IVF (26); the usual sperm dose is of the order of 1 million/ml. Penicillamine, hypotaurine and epinephrine (PHE) as a sperm motility stimulating mixture has been employed on the basis of data showing a f avourable effect (27). In the normal way, matured oocytes are partially stripped of cumulus cells by
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pipetting prior to exposure to sperm. A sodium citrate solution (3%) has been used to effectively 'clean' matured this adversely affecting oocytes without IVF either frequency or subsequent eatbryonic development; this may prove useful in enhancing the speed and reproducibility of oocyte penetration for IVF on an industrial scale (28). CULTURING THE EARLY_~O_~.IEIE",,EHBRYO.. The use of the rabbit oviduct as an effective in vivo culture system for the early bovine embryo was first shown by sreenan et al. (29). This in vivo system has been widely used by Canadian workers with IVF embryos derived from in vivo matured cattle eggs and by Japanese workers with in vitro matured and fertilized eggs to bring the zygote to the morula/blastocyst stage of development. In Dublin, the sheep rather than the rabbit has been favoured as the in vivo system. It is now clear that, when necessary several hundred cattle zygotes can be introduced into the ligated oviducts compromising percentage of without the eggs recovered(30). The usual routine has been to introduce eggs into the ligated oviduct a few hours after ovulation in the ewe, which is controlled by progestogen-PMSG treatment; the inent here is to provide eggs with much the same oviductal environment as in the live cow. Efficiency of egg recovery from the ewe is likely to vary widely according to animal of workers; factors and the experience for individual sheep, recovery rate can vary from nil to practically 108%. In terms of the vield of cattle embryos at the transferable stage of development, the initial Dublin results (4,5) in a to be difficult to research laboratory setting proved repeat under a semi-commercial production system. TABLE ~_--~ 2. SUMMARY OF RESULTS FOR YIELDS OF CATT&~_E,MBRY,O,S CULTURED IN VIVO IN THE SHEEP OVIDUCT _~._~e~o~~Ti-__._....-___.__.~.,_...,.__".~.-__.__.__.._ Oocytes Yxeld of Maturation Authors Oocytes cleaved embryos Details to ewe from ewe _______________________________~___~~~~______~___~~~_~~~~_~ Lu et al. 202 4(2.7%) 149 74%) 98 66%) FCS-c Hormones (L5) Lu et al. 119 78 66%) 23(29.5%) FCS+ 55 71%) Hormones (16) 86(60.0%) ecs+ Lu et al. 222 143 64%) 121 85%) Hormones (4) Lu et al. 668 420 63%) 334 80%) lPl(46.03) ecsHormones (51 UCD/ 18,895 7401 31,469 ECSOVAMASS 55,797 (56%) (60%) (23.5%) Data Hormones ______________________~~_____~~~_~~~~~~~~~~~~~~~~~~~~~~~~~~ Table 2 sets out data for the percentage of eggs which
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THERIOGENOLOGY showed evidence of cleavage when recovered from the sheep and the yield of embryos at the morula/blastocyst stage of development. However, a significant correlation (rr0.86) was found to exist in the UCD/Ovamass data between the cleavage rate of eggs recovered and the.yield of embryos. In examining data for the 156 groups of oocytes which went to make up that data, those groups that showed the highest cleavage rates (SS-90%) gave embryo yields averaging 43%, which is in broad agreement with the previous small-scale laboratory findings (4,s): however, this level of embryo yield was only achieved in 9% of the oocyte groups fsee Table 3). TABLE 3. YIELD OF EMSRYOS IN RELATXON TO CLEAVAGE RATE IN EGGS RECOVERED PROM THE SHEEP OVIDUCT (UCD/Ovanass Data)_ NO-Groups and as Cleavage Rates Yield of Embryos % of all Groups (mean) (range) __-___________"_____~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~~~ 14 (9.0%) 80.0--89.0% 42.8% 23 (14.7%) 70.0--79.9% 36.2% 31 (19.9%) 60.0--69.9% 25.2% 44 (28.2%) 50.0--59.9% 17.7% 40.0--49.9% 21 (13.5%) 15.6% I1 (7.1%) 30.0--39.9% 8.9% 7 (4.5%) 20.0--29.9% 6.1% less than 20% 5 (3.2%) 3.0%
The fact that even at the highest cleavage rates, no more than about 50% of eggs progress to the morula/blastocyst stage is a matter of obvious concern and requires much further research effort to identify the causes.
_r,2..._v_itro_~~b~c, For
the production of cattle embryos on an industrial scale, it is clearly essential that the sheep as an intermediate host should be phased out at the earliest opportunity. Although culture of IVF embryos to the morulafblastocyst stage in conventional culture media is not possible because of the block at the 8-16 cell stage, it was hoped that the oviductal cell monolayer technique might provide an effective answer to this problem. In Dublin, working with bovine oviductal monolayers, studies have shown M-199 to be superior to Ham's F-10 and ECS to be superior to FCS (31,32). It is also evident that the yield of embryos can be influenced by the concentration of bovine serum employed and by additives such as insulin (8). A strong relationship has been found to exist between the stage of development reached by the embryo at 48 h postinsemination and its subsequent progress to the morula/blastocyst stage (27); some further evidence of this is provided in Table 4. In the Dublin work, oviducts are obtained froar cattle showing evidence of recent ovulation.
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THERIOGENOLOGY TABLB 4.
EFFECT OR CELL STAGE AT TIME OF PLACEMENT ON BOVINE IIONOLAYRR ON YIELD OF EMBRYOS AT 7-DAYS.(Vergos et al.81 Cell stage No. Eggs put No. Reaching Embryo % Embryos stage f MorlBlast) on monolayer 6 0 -- 2 248 26 3-cell 139 47 g-cell 138 39 S-6 cell 71 Eggs placed on bovine oviductal monolayer
2.4% 18.7% 34.0% 54.9% at 48 h after IVF
Studies elsewhere shown that nonand in Dublin have oviductal cells, such as cumuluslgranulosa cells can be successfully used in a monolayer culture system to yield viable cattle embryos that they lag behind in vivo cultured embryos in their development;in some comparisons (27) they their in vivo appear to be a behind cultured day counterparts or embryos flushed from superovulated donors at a week of age. This would appear to be symptomatic of some defect in the monolayer system. Until it is clear what the monolayer systems lack that is supplied by the intact oviduct, there may be scope for employing interim measures such as the organ culture system described for growing hamster (35) and pig (36) eggs to the blastocyst stage in the isolated mouse oviduct. On the other hand, it may be a the matter examining range of factors of (cellular,endocrine) which are already known to affect the function of monolayer systems. The ultimate proof of an effective IVF system must always be in the birth of live calves and although there is good evidence of being able to achieve normal pregnancy rates with the in vivo culture methods used in Dublin, the numbers born after in vitro culture are more limited. Certainly,pregnancies can be established, but the embryo survival rate is an important factor in determining what can be done commercially with the embryos. There is also the question of embryo survival rate after standard freezethaw procedures; embryo quality and stage of development can place obvious limitations on what can be done in that area. More work is needed.
IN VITRO EHSRYOS ON THE FARM The development of effective non-surgical methods for the recovery and transfer of cattle embryos marked an important exploitation of cattle ET stage in the commercial technology. Dublin work in the mid-1970' was instrumental could be in showing that twin-pregnancies in cattle established in a reasonable percentage using the standard the Cassou transfer instrument. inseminating gun as However, the problem at that time was one of having a suitably low-cost supply of beef embryos. In the United Kingdom (UK), where there is some population of Holstein
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THERIOGENOLOGY
interest cattle, commercial using 3/4-bred may include continental sired embryos to produce high quality singleton twins. rather calves than Once low-cost sex predetermination methods can be grafted on to existing IVF technology, the birth of bull calves rather than heifers would have obvious advantages in a beef-rearing context. Twins by_g_mmgyo Transfer.: In seeking to establish twin-pregnancies, there is a choice of several approaches. The method developed in Ireland is one involving the deposition of a single embryo in the horn of the uterus contralateral to the ovulating ovary of a recipient cow which has bred at the previous heat. It has been shown that such a combined artificial insemination (AI) and embryo transfer approach is capable of achieving a twin-calving rate of about 50% in the cattle that become pregnant to first service (37). There is ample evidence in the literature suggesting that most natural twin-sets are carried in the form of bicornual twins. The evidence in several studies indicates that where the two foetuses are distributed between the two horns of the uterus, there is likely to be the advantage of a greater placental area. The cows twin-bearing is an important diagnosis of consideration from the farmer's viewpoint. This can be done around day-50 of pregnancy using real-time ultrasonics or by use of a total estrogen assay (6). The question with the day-50 diagnosis is in the loss of foetuses beyond that time and suffers from overlap between the estrogen levels cattle. twin-bearing Various and research in single centres, both in the Republic and the UK, are currently investigating in some depth the effects of pre-calving nutrition on the performance of in vitro produced embryos in establishing pregnancies in the cows; at Hillsborough cows Research Institute, work with 97 Friesian milking resulted in a pregnancy rate of 56% and a twining rate of 38%; there is much yet to be done in the refinement of field applications of the technology. Future
DeveloQments. The artificially matured cattle egg, produced by current are likely technology or modifications of that-technology to be of interest to those concerned with the development methods. An advantage cloning of such of large-scale cloning, once it is developed is the fact that it can be linked very readily to sexing: it becomes a matter of establishing the gender of a clone by conventional methods and this information relates to the others in the group. There is also likely to be growing commercial interest in the application of recombinant DNA technology in cattle, whether for improving animals for farming needs or for cows to produce unique for getting human proteins pharmacological purposes. In vitro production of embryos appears to have good potential and future uptake of the technology is likely to be significant.
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THERIOGENOLOGY REFERENCES 1.
Cheng, W.T.K., Moor, R.M. and Polge, C. In vitro fertilization of pig and sheep oocytes matured in vivo and in vitro. Theriogenology B:l46 (1986).
2.
Hanada, A., Enya, Y. and Suzuki, T. Birth of calves by nonsurgical transfer of in vitro fertilized embryos obtained from oocytes matured in vitro. Jap. J. Anim. Reprod. x:208 (1986).
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Mattioli, M., Bacci, M.L., Galeati, G. and Seren, E. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology =:1201 (1989).
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Lu, K.H., Gordon, I., Gallagher, M. and McGovern, H. Pregnancy established in cattle by transfer of embryos derived from IVF of oocytes matured in vitro. Vet. Rec. 1213259-260 (1989).
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Lu, K.H., Gordon, I., McGovern, H. and Gallagher, M. Production of cattle embryos by in vitro maturation and fertilization of follicular oocytes and their subsequent culture invivo in sheep. Theriogenology a:272 (1988).
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Lu, K.H., MaeDonnell, H.F. and Gordon, I. Birth of calves after in vitro maturation and fertilization of follicular oocytes. Theriogenology a:222 (1989).
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Yang, N.S., Lu, K.H. and Gordon, I. Invitro fertilization (IVF) of bovine oocytes from stored ovaries. Theriogenology 33 (1990).
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Vergos, E., Kinis, A., Gallagher, data) (1990).
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Yang, Y.B. and Lu, K.H. The influence of bovine oocytes type on in vitro fertilization and subsequent in vitro development. Theriogenology a (1990).
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Motlik, 3. and Fulka, 3. Factors affecting meiotic competence (1986). in pig oocytes. Theriogenology 25: 87-96
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Tan, S.J. and Lu, K.H. Effects of different cyclic stages of ovaries and sizes of follicles on development of IVF early bovine embryos. Theriogenology 22 (1990).
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Choudary, J.B., Gier, H.T. and Marion, G.B. Cyclic changes in bovine vesicular follicles. J. Anim. Sci. =:468-471 (1968).
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Lussier, J.G., Matton, P. and Dufour, J.J. Growth rates of follicles in the ovary of the cow. J. Reprod. Fert. &l-:301-307
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and Gordon, A.
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Staigmiller, R.B. and Moor, R.M. Effect of follicle cells on the maturation and developmental competence of oocytes matured outside the follicle. Gamete Res. 2:221-229 (1984).
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K.H., Boland, M.P., Crosby, T.F. and Gordon, I. III vitro fertilization of cattle oocytes matured in vitro. Theriogenology Z:25I (3.987).
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Lu, K.W., Gordon, I., Boland, M.P. and Crosby, T.F. In vitro fertilization of bovine oocytes. Brit, Sot. Anim. Prod. (Winter Mt), Paper 18 (1987).
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Lu, K.H. and Gordon, I. Effect of serum, hormones and cumulus cells on the in vitro maturation of bovine oocytes. Proc. Sot. Study Fertility & (1987).
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Leibfried-Rutledge, M.L., Critser, E.S., Parrish, J.J. and First, N.L. In vitro maturation and fertilization of bovine oocytes. Theriogenology 2:61-74 (1989).
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Sanbuissho, A. and Threlfall, W.R. The effects of estrous cow serum on the in vitro maturation and fertilization of the bovine follicular oocytes. Theriogenology 2:300-309 (1989).
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Younis, A.I., Brackett, B.G. and Fayrer-Hosken, R.A. Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro. Gamete Res. 2:189-201 (1989).
21.
Sirard, M.A., Parrish, J.J., Ware, C.B., Leibfried-Rutledge, M.L. and First, N.L. The culture of bovine oocytes to obtain developmentally competent embryos. Biol. Reprod. %:546-5X? (1988).
22.
Vergos, E., Kinis, A., Gallagher, M. and Gordon, A. Effect of oestradiol in maturation medium on development of the bovine embryo. Proc. 5th Mt. European Embryo Transfer Assoc. (Lyon) m (1989).
23.
Parrish, J.J., Parrish, J.L. and First, M.L. Effect of swim-up separation and heparin pretreatment of frozen-thawed spermatozoa on in vitro fertilization of bovine oocytes. Biol. Reprod. 30 (Suppl l):U (1984).
24.
Effect of pH on capacitation and Lu, K.H. and Gordon, I. fertilization medium on the in vitro fertilization of bovine oocytes. Proc. Sot. Study Fertility 12 (1987).
25.
Effect of bulls on Shi, D.S., Lu, K.H. and Gordon, I. fertilization of bovine oocytes and their subsequent development in vitro. Theriogenology 33 (1990).
26.
Ling, Z.J. and Lu, K.W. Frequency of cleavage and development in vitro of bovine oocytes fertilized in different number in drops with different sperm concentrations. Theriogenology &j (1990).
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27.
Vergos, E., Gordon, A., Gallagher, M. and Gordon, I. In vitro culture of embryos produced by in vitro maturation and IVF of bovine oocytes. Anim. Prod. &&:621 (1989).
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Kinis, A., Verges. E., Gallagher, M. and Gordon, A. Use of sodium citrate in the denudation of bovine oocytes. Proc. 5th Mt. European Embryo Transfer Assoc. (Lyon) 112 (1989).
29.
Sreenan, J., Scanlon, P.F. and Gordon, I. Culture of fertilized cattle eggs. J. Agric. Sci. (Camb.) =:183-185 (1968).
30.
Lu, K.H., Gordon, I., Gallagher, M. and McGovern, H. of cattle embryos by in vitro and in vivo culture. Anim. Prod. (Winter Mt.), Paper 53 (1988).
31.
Lu, K.H., Gordon, I., Chen, H.B., Gallagher, M. and McGovern, H. Birth of twins after transfer of cattle embryos produced by in vitro techniques. Vet. Rec. m:539-540 (1988).
32.
Wang, W.L., Jiang, H.S., Lu, K.H., McCarthy, D. and Gordon, I. The effect of media and sera on the in vitro development of early bovine embryos. J. Reprod. Fert. Abstract Series No. 3:50 (1989).
33.
Goto, K., Kajihara, Y., Kosaka, S., Koba, M., Nakanishi, Y. and Ogawa, K. Pregnancies after co-culture of cumulus cells with bovine embryos derived from in vitro fertilization of in vitro matured follicular oocytes. J. Reprod. Fert. Q:753-758 (1988).
34.
Jiang, H.S., Wang, W.L., Lu, K.H., McCarthy, D. and Gordon, I. In vitro culture of early bovine embryos derived from in vitro fertilization, comparison with invivo culture. J. Reprod. Fert. Abstract Series No. 3:50 (1989).
35.
Development of Minami, R., Bavister, B.D. and Iritani, A. hamster 2-cell embryos in the isolated mouse oviduct in organ culture system. Gamete Res. u:235 (1988).
36.
Krisher, R.L., Petters, R.M., Johnson, B.H., Bavister, B.D. and Archibong, A.E. Development of porcine embryos from one-cell to blastocyst in mouse oviducts maintained in organ culture. J. Exp. 2001. =:325-329 (1989).
37.
Experimental Diskin, M.G., McDonagh, T. and Sreenan, J. induction of twin-calving in beef cows by embryo transfer. Theriogenology a:224 (1987).
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PRODUCTION OF EHBRYOS IN VITRO AND ITS IMPACT ON LIVESTOCK PRODUCTION I. Gordon & K. H. Lul Department of Animal Science h Production, University College Dublin, Lyons Estate, Newcastle, County Dublin, Ireland ABSTRACT In Ireland, an attempt has been made to scale-up a laboratory procedure, which had yielded promising results, to meet the needs of extensive commercial exploitation of the technology of in vitro cattle embryo production. This paper examines some of the problems which have to be dealt with in bringing the embryo production technology to the point at which it becomes fully viable commercially. Much of the information included in the review is derived from on-going research and development work in the University; data are also derived from the work of Ovamass Ltd., a in 1987 to develop company established in Ireland the technology of embryo production on a commercial basis. Key words: cattle, in vitro, oocytes, embryo production.
young in limited numbers In recent years, have been produced in sheep (11, cattle (2) and pigs (3) by (IVF) of techniques involving the in vitro fertilization ovarian oocytes recovered from slaughterhouse animals and matured in vitro. Of the farm animals, it is in cattle that interest lies in greatest getting the the emerging technology of in vitro embryo production developed to the of commercial relevance. point of being Although nonmethods for surgical recovery obtaining embryos from superovulated donor cattle have been available for some are costly. such methods The ovaries of years, slaughterhouse cattle, on the other hand, could provide an appropriately low cost supply of oocytes that could be and cultured in fertilized vitro to the matured, transferable stage of development. In Dublin, a production system has been devised to provide such a low cost supply of cattle embryos using slaughterhouse cattle ovaries as the raw materials (4,5,6) and this has resulted in attempts to commercialise the procedure and through embryo transfer to apply the new technology on the farm. 1. Present Address: Ovamass Ltd.,Fethard, County Tipperary. Acknowledgements: The support of Ovamass Ltd is gratefully acknowledged.
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However, it is now evident that further developments and are required in bringing refinements embryo production technology to the point at which it can have significant commercial impact. Although oocytes in large numbers can be recovered from slaughterhouse ovaries, there are problems yet to be overcome in consistently producing transferable/ freezable quality embryos in quantity. For thought of any extensive application of non-surgical embryo transfer on the farm, there are several considerations relating to such techniques and the most appropriate transfer ways of husbandry problems dealing with the that arise, may particularly when the application is directed towards the induction of twin-calvings. PRODUCING EMBRYOS IN THE_LABORATOm Collection ofOvaries.‘----‘--The raw rnaterialszsoyed in the large-scale production of cattle embryos have been the ovaries of slaughterhouse heifer cattle. The original protocol involved bringing the ovaries from the abattoir in medium held at 30-37°C (1) but is now clear that the ovaries can be held at 200C for at oocytes are recovered without least 8 h before this compromising the acceptability of oocytes for maturation and fertilization (7). This permits much greater freedom in arranging production line work. Each bovine ovary contains many thousands of oocytes, but with current technology only a relatively small proportion follicle population is utilized. of the total vesicular dissection was initially employed in Although follicle reccvering oocytes from follicles (2-6 mm diameter), for routine use, aspiration is now the method of choice. The obvious advantage of aspiration iS in terms of speed of important in an embryo operation, which is especially production line system. Data in Table 1 show that the outcome at 48 h post-insemination, was not compromised by using aspiration rather than follicle dissection (8). COMPARISON OF FOLLICLE DISSECTION AND ASPIRATION TABLE 1. 4S METHODS OP OOCYTB RBC?~~_EMPLO_YED_IN.C_~_TTLE_IVF~,_ +--.--. Me t h o d Dissection Aspiration
Ovaries 892 646
Oocytes Recovered 5822 4511
Oocytes Cultured 3642 3310
Put to IVF
Cleavage Rate'
2974 2494
83.5% 82.4%
The principle of maintaining an intact cumulus-oocytecomplex (C-O-C) is regarded as an important one to observe at all times and evidence is available that this can have a genuine effect on the yield of embryos (9).
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The maximum number of usable oocytes recoverable per ovary, using follicles of 2 mm diameter and above, would be about 10 (8). Reports in the literature generally show some 40-50 vesicular follicles to be in the 2mm and above category but only about 50% of these would be capable of yielding an acceptable There is need oocyte. for much further information on the incidence and extent of follicular atresia in relation to follicle size and the reproductive status of the cattle that are being used as the source of ovaries. Cow ovaries have been found to yield a smaller number of acceptable oocytes than beef heifer cattle that are slaughtered at 2-3 years (8). There is also the question of the meiotic competence of oocytes in follicles that are one mm diameter and less in diameter. The data of Notlik 61 Fulka (10) indicate that bovine follicles up to 1.6 mm diameter may contain oocytes that are still growing and have not yet attained the state of being meiotically competent.Studies conducted in Ovamass by Tan & Lu (11) have shown that oocytes recovered from give follicles below 2mm diameter significantly lower yields of embryos after in vitro maturation and IVF. There is a need to examine procedures whereby growing oocytes obtained from the small follicles can be cultured to the point at which they do attain meiotic competence. In terms of the total vesicular follicle population in the bovine numbers present there are large the ovary, (12,13); challenge is one of devising appropriate ways of fully utilizing the oocytes which the contain. OOCYTE MATURATION.. In sheep. the requirement for follicle cell support in the Cf the oocyte was cytoplasmic maturation clearly demonstrated (14) and this same principle was applied in the initial Dublin work, using additional cumulus cells and hormone supplementation (15). This protocol was modified to exclude hormones on the basis of data from subsequent studies (16,17). In using additional cumulus cells in the current production system, there is no evidence of any temporary inhibition of meiosis in the bovine oocytes, such as that recorded by some workers (18) when cumulus cells have been added to culture medium prior to commitment of the oocytes to germinal vesicle break-down. A non-static routinely employed as a means of system is culture preventing the plating of cumulus cells and incubations are carried out at 39'C, the core temperature of the cow. (H-199) is currently supplemented The maturation medium with 20% estrous cow serum (ECS),this form of serum being employed rather than fetal calf serum (FCS) on the basis of earlier work (16).Subsequent results have not always shown similar evidence of an advantage(E).Elsewhere,studies have shown ECS to have a favourable effect on IVF in comparison with sera obtained at ovulation and 24 h after ovulation
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(19) i others have reported pro-estrous serum to have the most favourable effect on IVF in their studies.There are obviously steroids,peptide hormones and growth factors in bovine serum whose role in oocyte maturation remains to be determined. Medium 199 is generally employed in maturing bovine oocytes and is usually supplemented with gonadotropins (FSH/LH) and estradiol; in sheep, such supplementation had proved to be essential in cattle some work has shown a (14) and favourable effect (20,21).In Dublin, although oocytes are routinely matured in the absence of hormones, there is some effect of estradiol evidence of a favourable in the maturation medium which becomes evident at the 7-day stage of development (22). There is clearly a need for much further work in examining the hormonal requirements of oocyte maturation in the cow, since present evidence is somewhat conflicting. In Dublin, immature eggs are cultured for 24-26 h in groups of 40 in 2 ml volumes of maturation medium. The routine maturation procedure has been shown to be capable of yielding more than 90% of oocytes at metaphase II (17). SPERM CAPACITATIOW, For an effective IVF system,it is clearly essential to have a reliable means of preparing sperm. In Dublin, although several procedures for capacitating bull sperm have been examined, the most effective to date is the method based on that reported by Parrish et al. (23) who demonstrated that heparin could increase IVF frequencies: details of the heparin treatment as used in Dublin have been reported elsewhere used in (15,24). The semen routine embryo production is obtained from local cattle breeding stations and semen straws from several bulls within the selected breed are pooled to provide sperm for the IVF. A 60-minute swim-up procedure is used to isolate a highly motile sperm population prior to washing and a 15 min. incubation period with heparin. In terms of factors affecting the outcome of of difficulty in IVF, bull variability has been one source the Dublin work, with certain individual bulls or even bulls groups of within a breed showing much higher fertilization frequencies than others (25). IN VITRO FERTILIZATThe fertilization of matured oocytes has been routinely carried out in microdroplets of HEPES-TAPES medium droplets contain 10 normally oocytes but (pH,7.S); considerably larger numbers (UP to 50) can be included without compromising the outcome of IVF (26); the usual sperm dose is of the order of 1 million/ml. Penicillamine, hypotaurine and epinephrine (PHE) as a sperm motility stimulating mixture has been employed on the basis of data showing a f avourable effect (27). In the normal way, matured oocytes are partially stripped of cumulus cells by
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pipetting prior to exposure to sperm. A sodium citrate solution (3%) has been used to effectively 'clean' matured this adversely affecting oocytes without IVF either frequency or subsequent eatbryonic development; this may prove useful in enhancing the speed and reproducibility of oocyte penetration for IVF on an industrial scale (28). CULTURING THE EARLY_~O_~.IEIE",,EHBRYO.. The use of the rabbit oviduct as an effective in vivo culture system for the early bovine embryo was first shown by sreenan et al. (29). This in vivo system has been widely used by Canadian workers with IVF embryos derived from in vivo matured cattle eggs and by Japanese workers with in vitro matured and fertilized eggs to bring the zygote to the morula/blastocyst stage of development. In Dublin, the sheep rather than the rabbit has been favoured as the in vivo system. It is now clear that, when necessary several hundred cattle zygotes can be introduced into the ligated oviducts compromising percentage of without the eggs recovered(30). The usual routine has been to introduce eggs into the ligated oviduct a few hours after ovulation in the ewe, which is controlled by progestogen-PMSG treatment; the inent here is to provide eggs with much the same oviductal environment as in the live cow. Efficiency of egg recovery from the ewe is likely to vary widely according to animal of workers; factors and the experience for individual sheep, recovery rate can vary from nil to practically 108%. In terms of the vield of cattle embryos at the transferable stage of development, the initial Dublin results (4,5) in a to be difficult to research laboratory setting proved repeat under a semi-commercial production system. TABLE ~_--~ 2. SUMMARY OF RESULTS FOR YIELDS OF CATT&~_E,MBRY,O,S CULTURED IN VIVO IN THE SHEEP OVIDUCT _~._~e~o~~Ti-__._....-___.__.~.,_...,.__".~.-__.__.__.._ Oocytes Yxeld of Maturation Authors Oocytes cleaved embryos Details to ewe from ewe _______________________________~___~~~~______~___~~~_~~~~_~ Lu et al. 202 4(2.7%) 149 74%) 98 66%) FCS-c Hormones (L5) Lu et al. 119 78 66%) 23(29.5%) FCS+ 55 71%) Hormones (16) 86(60.0%) ecs+ Lu et al. 222 143 64%) 121 85%) Hormones (4) Lu et al. 668 420 63%) 334 80%) lPl(46.03) ecsHormones (51 UCD/ 18,895 7401 31,469 ECSOVAMASS 55,797 (56%) (60%) (23.5%) Data Hormones ______________________~~_____~~~_~~~~~~~~~~~~~~~~~~~~~~~~~~ Table 2 sets out data for the percentage of eggs which
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THERIOGENOLOGY showed evidence of cleavage when recovered from the sheep and the yield of embryos at the morula/blastocyst stage of development. However, a significant correlation (rr0.86) was found to exist in the UCD/Ovamass data between the cleavage rate of eggs recovered and the.yield of embryos. In examining data for the 156 groups of oocytes which went to make up that data, those groups that showed the highest cleavage rates (SS-90%) gave embryo yields averaging 43%, which is in broad agreement with the previous small-scale laboratory findings (4,s): however, this level of embryo yield was only achieved in 9% of the oocyte groups fsee Table 3). TABLE 3. YIELD OF EMSRYOS IN RELATXON TO CLEAVAGE RATE IN EGGS RECOVERED PROM THE SHEEP OVIDUCT (UCD/Ovanass Data)_ NO-Groups and as Cleavage Rates Yield of Embryos % of all Groups (mean) (range) __-___________"_____~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~~~ 14 (9.0%) 80.0--89.0% 42.8% 23 (14.7%) 70.0--79.9% 36.2% 31 (19.9%) 60.0--69.9% 25.2% 44 (28.2%) 50.0--59.9% 17.7% 40.0--49.9% 21 (13.5%) 15.6% I1 (7.1%) 30.0--39.9% 8.9% 7 (4.5%) 20.0--29.9% 6.1% less than 20% 5 (3.2%) 3.0%
The fact that even at the highest cleavage rates, no more than about 50% of eggs progress to the morula/blastocyst stage is a matter of obvious concern and requires much further research effort to identify the causes.
_r,2..._v_itro_~~b~c, For
the production of cattle embryos on an industrial scale, it is clearly essential that the sheep as an intermediate host should be phased out at the earliest opportunity. Although culture of IVF embryos to the morulafblastocyst stage in conventional culture media is not possible because of the block at the 8-16 cell stage, it was hoped that the oviductal cell monolayer technique might provide an effective answer to this problem. In Dublin, working with bovine oviductal monolayers, studies have shown M-199 to be superior to Ham's F-10 and ECS to be superior to FCS (31,32). It is also evident that the yield of embryos can be influenced by the concentration of bovine serum employed and by additives such as insulin (8). A strong relationship has been found to exist between the stage of development reached by the embryo at 48 h postinsemination and its subsequent progress to the morula/blastocyst stage (27); some further evidence of this is provided in Table 4. In the Dublin work, oviducts are obtained froar cattle showing evidence of recent ovulation.
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THERIOGENOLOGY TABLB 4.
EFFECT OR CELL STAGE AT TIME OF PLACEMENT ON BOVINE IIONOLAYRR ON YIELD OF EMBRYOS AT 7-DAYS.(Vergos et al.81 Cell stage No. Eggs put No. Reaching Embryo % Embryos stage f MorlBlast) on monolayer 6 0 -- 2 248 26 3-cell 139 47 g-cell 138 39 S-6 cell 71 Eggs placed on bovine oviductal monolayer
2.4% 18.7% 34.0% 54.9% at 48 h after IVF
Studies elsewhere shown that nonand in Dublin have oviductal cells, such as cumuluslgranulosa cells can be successfully used in a monolayer culture system to yield viable cattle embryos that they lag behind in vivo cultured embryos in their development;in some comparisons (27) they their in vivo appear to be a behind cultured day counterparts or embryos flushed from superovulated donors at a week of age. This would appear to be symptomatic of some defect in the monolayer system. Until it is clear what the monolayer systems lack that is supplied by the intact oviduct, there may be scope for employing interim measures such as the organ culture system described for growing hamster (35) and pig (36) eggs to the blastocyst stage in the isolated mouse oviduct. On the other hand, it may be a the matter examining range of factors of (cellular,endocrine) which are already known to affect the function of monolayer systems. The ultimate proof of an effective IVF system must always be in the birth of live calves and although there is good evidence of being able to achieve normal pregnancy rates with the in vivo culture methods used in Dublin, the numbers born after in vitro culture are more limited. Certainly,pregnancies can be established, but the embryo survival rate is an important factor in determining what can be done commercially with the embryos. There is also the question of embryo survival rate after standard freezethaw procedures; embryo quality and stage of development can place obvious limitations on what can be done in that area. More work is needed.
IN VITRO EHSRYOS ON THE FARM The development of effective non-surgical methods for the recovery and transfer of cattle embryos marked an important exploitation of cattle ET stage in the commercial technology. Dublin work in the mid-1970' was instrumental could be in showing that twin-pregnancies in cattle established in a reasonable percentage using the standard the Cassou transfer instrument. inseminating gun as However, the problem at that time was one of having a suitably low-cost supply of beef embryos. In the United Kingdom (UK), where there is some population of Holstein
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THERIOGENOLOGY
interest cattle, commercial using 3/4-bred may include continental sired embryos to produce high quality singleton twins. rather calves than Once low-cost sex predetermination methods can be grafted on to existing IVF technology, the birth of bull calves rather than heifers would have obvious advantages in a beef-rearing context. Twins by_g_mmgyo Transfer.: In seeking to establish twin-pregnancies, there is a choice of several approaches. The method developed in Ireland is one involving the deposition of a single embryo in the horn of the uterus contralateral to the ovulating ovary of a recipient cow which has bred at the previous heat. It has been shown that such a combined artificial insemination (AI) and embryo transfer approach is capable of achieving a twin-calving rate of about 50% in the cattle that become pregnant to first service (37). There is ample evidence in the literature suggesting that most natural twin-sets are carried in the form of bicornual twins. The evidence in several studies indicates that where the two foetuses are distributed between the two horns of the uterus, there is likely to be the advantage of a greater placental area. The cows twin-bearing is an important diagnosis of consideration from the farmer's viewpoint. This can be done around day-50 of pregnancy using real-time ultrasonics or by use of a total estrogen assay (6). The question with the day-50 diagnosis is in the loss of foetuses beyond that time and suffers from overlap between the estrogen levels cattle. twin-bearing Various and research in single centres, both in the Republic and the UK, are currently investigating in some depth the effects of pre-calving nutrition on the performance of in vitro produced embryos in establishing pregnancies in the cows; at Hillsborough cows Research Institute, work with 97 Friesian milking resulted in a pregnancy rate of 56% and a twining rate of 38%; there is much yet to be done in the refinement of field applications of the technology. Future
DeveloQments. The artificially matured cattle egg, produced by current are likely technology or modifications of that-technology to be of interest to those concerned with the development methods. An advantage cloning of such of large-scale cloning, once it is developed is the fact that it can be linked very readily to sexing: it becomes a matter of establishing the gender of a clone by conventional methods and this information relates to the others in the group. There is also likely to be growing commercial interest in the application of recombinant DNA technology in cattle, whether for improving animals for farming needs or for cows to produce unique for getting human proteins pharmacological purposes. In vitro production of embryos appears to have good potential and future uptake of the technology is likely to be significant.
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1990VOL. 33 NO. 1
THERIOGENOLOGY REFERENCES 1.
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