Temporary Exposure of Ovine Embryos to an Advanced Uterine Environment Does Not Affect Fetal Weight but Alters Fetal Muscle Development¹

^c Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB, Scotland, United Kingdom ^d The Scottish Agricultural College, Craibstone, Bucksburn, Aberdeen, AB21 9SB Scotland, United Kingdom

	ABSTRACT

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Embryo transfer techniques may result in fetuses that are heavier at birth and that have been described as highly muscled. The aim of this study was to investigate myogenesis in lambs derived from embryo transfer. Embryos were transferred at Day 3 (estrus = Day 0) to a 3 days-advanced uterine environment, maintained there for 3 days, recovered, and then returned to a synchronous (Day 6) uterus; these fetuses comprised the asynchronous group. Control animals were created by synchronous embryo recovery and single transfer at Day 3. Asynchronous transfer did not affect fetal weight or curved crown-rump length between 46 and 135 days of gestation. No differences were detected between groups at Days 110–135 with respect to muscle mass or protein, RNA, and DNA content. However, total muscle fiber number was significantly increased in plantaris muscles from the asynchronous groups at Day 110 and Day 125, suggestive of prolonged hyperplasia. In addition, the levels of Myf 5 protein and the secondary-to-primary fiber ratio were altered in plantaris muscle from the asynchronous group.

The growth data are in contrast to previously reported findings. The results show that fetal myogenesis can be altered by very early events in embryogenesis and suggest that any inferences made solely on the basis of fetal or muscle weight may be fallacious.

	INTRODUCTION

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The use of advanced reproductive technologies in domestic animal husbandry has led to the occurrence of abnormally large offspring [1]. For example, maternal administration of progesterone in the very early stages of pregnancy [2], nuclear cloning of embryos [3], and in vitro culture [4, 5] and coculture of embryos on a granulosa cell layer [6–8] all seem to affect fetal growth. In addition, the transfer of embryos to a temporary advanced uterine environment (asynchronous transfer) has been shown to increase crown-to-rump length by approximately 40% at 37 days of gestation [9] or to increase weight by 42% at 21 days of gestation [10]. Although the underlying mechanisms are unknown, the etiology of this phenomenon appears to be related to modifications in the overall embryonic or fetal growth curve in response to an abnormal early embryonic environment.

In commercial practice, field reports of "oversize" ruminant offspring have described them as highly muscled and stocky. This raises the question whether the phenomenon may involve some alteration in muscle development, since the relative degree of muscle cell proliferation and differentiation during fetal development appears to determine muscle mass at birth [11]. In addition, it has been suggested that muscle fiber number is an indication of fetal growth rate [12, 13]. Hence it might be speculated that fiber number would be increased in animals that were larger than normal at birth.

The objective of this study was therefore to determine whether embryo transfer to an advanced uterine environment affects myogenesis.

	MATERIALS AND METHODS

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The methodology employed for embryo recovery and transfer was based on that described previously [9]. Mature Scottish Blackface ewes (n = 252) were randomly assigned to be either embryo donors (n = 49), temporary recipients (n = 13), or permanent recipients (n = 190). In the experiment that followed, Day 3 embryos were recovered from superovulated donor ewes and transferred either synchronously to Day 3 (estrus = Day 0) permanent recipients or asynchronously to Day 6 temporary recipients. Asynchronously transferred embryos were recovered 3 days later and transferred to Day 6 permanent recipients. Gravid uteri were recovered subsequently on Days 35, 45, 60, 90, 110, 125, and 135 of gestation. The experiment therefore consisted of two treatments (synchronous vs. asynchronous embryo transfer) and seven recovery points (from Day 35 to Day 135 of gestation inclusive). The animal experiments were carried out in accordance with the Home Office Animals (Scientific Procedures) Act 1986 and in line with the Guiding Principles for the Care and Use of Research Animals.

Estrous cycles were synchronized for both donor and recipient ewes using progestogen-releasing intravaginal sponges (30 mg fluorogestone acetate [Chronogest]; Intervet, Cambridge, UK) for 12 days. In recipient ewes the same sponge remained in position for 12 days, whereas in donor ewes it was removed on Day 7 and replaced with a new one. Temporary and permanent recipients received, in addition, 400 IU eCG (Intervet) at sponge withdrawal.

Superovulation was induced in donor ewes using a total dose of 9 mg ovine FSH (Ovagen; ICP Ltd., Auckland, New Zealand) administered twice daily in equal doses over 4 days beginning on Day 10 of the 12-day progestogen-priming period. Laparoscopic intrauterine insemination was carried out 44 h after sponge withdrawal using fresh semen from a single Suffolk sire. Embryos were recovered from 49 ewes by laparotomy 3 days after insemination (Day 0) and transferred laparoscopically to synchronous (Day 3) permanent recipients until fetal recovery or to asynchronous (Day 6) temporary recipients for 3 days. Embryos were then recovered from these temporary recipients and transferred to Day 6 permanent recipients.

All embryos were recovered by laparotomy under general anesthesia (halothane, oxygen, and nitrous oxide). Day 3 embryos (Day 0 = insemination) were recovered by retrograde flushing of the uterine horn and oviduct, and Day 6 embryos by flushing from the uterotubal junction, down the uterine horn. After assessment of developmental stage, embryos were transferred either singly to permanent recipients or as multiples to temporary recipients. The transfer technique involved temporary exposure of the tip of the uterine horn through a small ventral incision, following its laparoscopically guided pickup.

Temporary recipients were brought into estrus 78 h earlier on average than donor ewes. The embryos that they received were maintained in vitro in ovum culture medium (Imperial Laboratories, Andover, UK) at 20–25°C for around 5 h before transfer. Permanent recipients came into estrus 2 h earlier on average than donor ewes, and the embryos they received were maintained under the same in vitro conditions as those for the temporary recipients but for around 3 h before transfer.

Pregnancy rates averaged 65% and were not different for the two treatment groups. In all, 121 fetuses were recovered (comprising 56 synchronous and 65 asynchronously derived animals). Fetal recovery followed the killing of pregnant recipient ewes, which was performed by intravenously administering 25 ml of a 20% (w:v) solution of pentobarbitone sodium (Euthatal; Rhône Mérieux Ltd., Harlaw, UK). The gravid uterus of each ewe was recovered several minutes after exsanguination; each fetus was recovered and weighed, and curved crown-rump length was measured.

Muscle dissection was not possible at Day 35 of gestation and so was restricted to Day 45 onward. However, at Days 45 and 60 it was not possible to dissect individual hind limb muscles with sufficient accuracy; hence the group of hind limb muscles comprising plantaris and medial and lateral gastrocnemius was removed and snap frozen for biochemical and Western analysis. At Days 110–135, individual plantaris and gastrocnemius muscles were excised and weighed, and a small sample from the midbelly of the muscle was taken for histochemistry. The remaining muscle tissue was snap frozen for biochemical analysis of protein, RNA, and DNA content and for Western analysis. The 10-µm cryostat transverse sections of whole muscle were cut and reacted for Ca²⁺-activated myofibrillar ATPase activity (E.C. 3,6,3,3) at pH 9.4 [14].

Total fiber number in plantaris muscle was estimated in sections stained for ATPase activity by semiautomatic image analysis (Leica Q600; Leica Ltd., Milton Keynes, UK). The total number of myofibers was counted in one transverse muscle section from each fetus. It was not possible to maintain the integrity of the whole muscle sections from the fetuses at 135 days; hence no total fiber counts were possible for this gestational age.

The ratio of secondary to primary fibers (S:P) was also estimated in sections of plantaris muscle from animals at 125 days of gestation. S:P was estimated by counting each primary and secondary fiber in 5 randomly chosen fields of known area. The mean of the ratios was taken to be the S:P.

Frozen muscles were pulverized, and total protein, RNA, and DNA were estimated using the methods previously described [15–17]. Aliquots of pulverized muscle (grouped hind limb muscle, Days 45 and 60; plantaris muscle, Days 90, 110, 125, and 135) were homogenized in ice-cold extraction buffer (20 mM Tris-HCl, 0.25 M sucrose, 10 mM EGTA, 2 mM EDTA, pH 7.5) with protease inhibitors (10 µl/ml PMSF, 30 µl/ml aprotonin, 10 µl/ml sodium orthovanadate) and then left on ice for 1 h, prior to centrifugation for 1 h (100 000 x g, 4°C). Then 0.5 ml of the extraction buffer containing 1% Triton X-100 was added to the pellet, which was rehomogenized, placed on ice for 1 h, and centrifuged for 30 min. Aliquots of the supernatant containing 40 µg protein were run on a 4.5% stacking and 10% separation SDS-polyacrylamide gel. Electrophoretically separated proteins were transferred to Hybond ECL (Amersham, Little Chalfont, UK) nitrocellulose membrane from SDS gels by electroblotting (Biometra, Gottingen, Germany) at 700 mA for 30 min. Western analysis was then performed using a rabbit polyclonal primary antibody (Myf 5; Santacruz Biotechnology Inc., Santa Cruz, CA) at 1:1000 followed by secondary antibody, rabbit immunoglobulin horseradish peroxidase-linked F(ab)₂ fragment from sheep (Amersham) at 1:2000 dilution. The binding was visualized by electrochemiluminescence.

Data were subjected to a Student's t-test with significance being assigned to values of p < 0.05.

	RESULTS

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As reported previously [18], asynchronous transfer had no effect on fetal growth. In addition, curved crown-rump lengths were not different at any points in gestation, nor was there any impact on the weight of the individual muscles at Days 90–135 of gestation (Table 1). Similarly, with the exception of DNA values at Day 125, asynchronous transfer did not affect total protein, RNA, and DNA contents in plantaris muscles obtained from Day 110, 125, and 135 fetuses (Table 2). The apparent difference in DNA values at Day 125 was significant at the 5% level, and in view of the lack of difference at any other time point, it is likely that this difference arose by chance.

However, observation of the transverse sections of plantaris strongly suggested that there was a difference in the number of muscle fibers present between the two treatment groups (Fig. 1). This was confirmed when the muscle fiber numbers were determined and results showed that at both Day 110 and 125 of gestation, total fiber number was significantly increased (p < 0.01 and p < 0.001, respectively) in plantaris muscles from fetuses derived by asynchronous transfer (Fig. 2). In addition, estimation of the S:P for animals at Day 125 showed that there was a significant (p < 0.001) increase in the ratio in the asynchronous group (8.80 ± 0.5; mean ± SD) as compared to the synchronous group (6.9 ± 0.4; mean ± SD).

View larger version (147K): [in this window] [in a new window]   FIG. 1. Transverse sections through ovine fetal plantaris muscle reacted to demonstrate the activity of Ca2+-activated ATPase. a) Day 110 gestation fetus from synchronous transfer, b) Day 125 fetus from synchronous transfer, c) Day 110 gestation fetus from asynchronous transfer, d) Day 125 fetus from asynchronous transfer. Note the difference in both fiber density (number) and size between treatments for both gestational time points.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Total muscle fiber number in ovine fetal plantaris muscles at Days 110 and 125 of gestation. Data from animals derived by synchronous transfer are represented by open bars; those from animals derived by asynchronous transfer are represented by solid bars. The data are means with SD; n = 6 for each group. **p < 0.01.

Data on the relative levels of Myf 5 protein in muscle at Days 45, 60, 90, 110, and 125 of gestation are given in Figure 3. For both treatment groups, the signal reduced with time, and for the asynchronous group, there was a particularly sharp reduction in Myf 5 levels at Day 90.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Examples of typical images from Western ECL analysis of the levels of Myf 5 protein in ovine fetal plantaris muscle from animals derived by synchronous or asynchronous transfer, recovered at 45, 60, 90, 110, and 125 days of gestation.

	DISCUSSION

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Previous reports of temporary exposure of ovine embryos to an advanced (asynchronous) uterine environment have shown growth changes at early gestational stages: an increased crown-rump length at Day 37 [9] and an increased fetal weight at Day 21 [10]. However, the current study was unable to replicate previous findings, and indeed there was no evidence that asynchronous transfer was associated with increased fetal growth at any stage during gestation. The reason for these differences is not clear, but it must be acknowledged that the present experimental design does not rule out the possibility that the effects observed are attributable to double transfer rather than the asynchronous environment.

Despite the lack of impact on muscle weight, the present data showed that muscle fiber number was significantly increased at Days 110 and 125 of gestation in the asynchronously derived fetuses. Evidence suggests that, independent of birth weight, muscle fiber number may be related to body size and postnatal growth potential [12] and that through this relationship with growth rate, muscle fiber number may provide a better indication of growth potential than either birth weight or crown-rump length [13]. Applying this concept to the present study would suggest that although fetal weight up to 135 days gestation was unaffected by asynchronous transfer, the observed increase in fiber number at Days 110 and 125 of gestation may have implications for future neonatal and/or postnatal growth.

The mechanism through which fiber number is increased is not clear, but the observed temporal difference in Myf 5 expression and the increase in S:P suggest that asynchronous transfer of embryos results in a prolongation of myoblast hyperplasia. In cattle [19] it appears that Myf 5 transcript levels change in a biphasic manner during development, peak levels possibly signifying particular stages in primary and secondary myogenesis such that transcript levels are high in myoblasts and fall with the switch from proliferation to fusion and differentiation. The elevated S:P in the asynchronous group would indicate either a reduction in primary fibers or an increase in secondary fiber numbers, but in light of the increase in total fiber number it is unlikely that primary fiber numbers are reduced. In addition, the elevated levels of Myf 5 at Day 110 would support the contention of extended hyperplastic growth in the secondary fiber population in the muscles of fetuses derived by asynchronous transfer.

In conclusion, asynchronous transfer of ovine embryos did not lead to increased fetal weight, size, or muscle weight from Day 35 to 135 gestation. However, the data suggest that manipulation of the preimplantation embryo can affect subsequent myogenesis in fetal muscle, although the underlying mechanism is not clear. Hence, by subjecting embryos to unusual environments it may be possible to affect the fetal muscle growth of animals with potential consequences for commercial livestock production.

	ACKNOWLEDGMENTS

 
Thanks are given to Drs. Ian Wilmut and Lorraine Young of Roslin Institute, Edinburgh, for fruitful discussions.

	FOOTNOTES

 
¹ This work was supported by the Scottish Office Agriculture, Environment and Fisheries Department and the Ministry of Agriculture Food and Fisheries.

² Correspondence: C.A. Maltin, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, Scotland, UK. FAX: 01224–716687; c.maltin{at}rri.sari.ac.uk

Accepted: March 18, 1998.

Received: September 23, 1997.

	REFERENCES

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1. Kruip ThAM, den Daas JHG. In vitro produced and cloned embryos: effects on pregnancy, parturition and offspring. Theriogenology 1997; 47:43–52.
2. Kleeman DO, Walker SK, Seamark RF. Enhanced foetal growth in sheep administered progesterone during the first three days of pregnancy. J Reprod Fertil 1994; 102:411–417.[Abstract]
3. Willadsen SM, Janzen RE, McAlister RJ, Shea BF, Hamilton G, McDermand D. The viability of late morulae and blastocysts produced by nuclear transplantation in cattle. Theriogenology 1991; 35:161–172.[CrossRef]
4. Walker SK, Heard TM, Seamark RF. In vitro culture of embryos without co-culture: successes and perspectives. Theriogenology 1992; 37:111–126.[CrossRef]
5. Holm P, Walker SK, Petersen BA, Ashman RJ, Seamark RF. In vitro vs in vivo culture of ovine IVM-IVF ova: effect on lambing. Theriogenology 1994; 41:217 (abstract).[CrossRef]
6. Farin PW, Farin CE, Yang L. In vitro production is associated with altered foetal development. Theriogenology 1994; 41:193 (abstract).[CrossRef]
7. Bernardi ML, Delouis C. Coculture of ovine zygotes fertilised in vivo or in vitro and positive effect of CZB medium on the development of in vitro fertilised zygotes. Reprod Nutr Dev 1995; 35:451–464.
8. Gandolfi F, Moor RM. Stimulation of early embryonic development in the sheep by coculture with oviductal cells. J Reprod Fertil 1989; 81:23–28.
9. Wilmut I, Sales DI. Effect of an asynchronous environment on embryonic development in sheep. J Reprod Fertil 1981; 61:179–184.[Abstract]
10. Young LE, Butterwith SC, Wilmut I. Increased ovine foetal weight following transient asynchronous embryo transfer is not associated with increased placental weight at day 21 of gestation. Theriogenology 1996; 45:231 (abstract).[CrossRef]
11. Handel SE, Stickland NC. Muscle cellularity and birth weight. Anim Prod 1987; 44:311–317.
12. Dwyer CM, Fletcher JM, Stickland NC. Muscle cellularity and postnatal growth in the pig. J Anim Sci 1993; 71:3339–3343.[Abstract]
13. Gore MT, Young RB, Claeys MC, Chromiak JA, Rahe CH, Marple DN, Hough JD, Griffin JL, Mulvaney DR. Growth and development of bovine foetuses and neonates representing three genotypes. J Anim Sci 1994; 72:2307–2318.[Abstract]
14. Hayashi M, Freiman DG. An improved method of fixation for formalin sensitive enzymes, with special reference to myosin adenosine triphosphatase. Histochem Cytochem 1966; 4:577–581.
15. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with folin reagent. J Biol Chem 1951; 193:265–275.[Free Full Text]
16. Munro HN, Fleck A. The determination of nucleic acids. Methods Biochem Anal 1967; 14:113–165.
17. Burton K. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956; 62:315–323.[Medline]
18. Sinclair KD, Tregaskes LD, Maxfield EK, Robinson JJ, Broadbent PJ, Maltin CA. Fetal growth following temporary exposure of day 3 ovine embryos to an advanced uterine environment. Theriogenology 1996; 45:223 (abstract).[CrossRef]
19. Kelley RL, Mulvaney DR. Developmental expression pattern of myogenic regulatory genes, MyoD, Myf-5 and in bovine skeletal muscle. J Anim Sci 1992; 70(suppl 1):10.