Pregnancy rates after transfer of in vitro produced (IVP) embryos: effects of epidermal

growth factor (EGF)

 

J.T. Choi, L.P. Reynolds, D.A. Redmer , J.S. Luther , C. Navanukraw, D. Pant, R.M. Weigl,

J.D. Kirsch, K.C. Kraft and A.T. Grazul-Bilska

 

Department of Animal and Range Sciences, North Dakota State University, Fargo

 

 

INTRODUCTION

 

The first sheep embryo transfer was reported about 70 years ago (Warwick et al., 1934).  However, most of the embryo transfer technology has been developed within the last 20 years (Ishwar and Memon, 1996). The advantages of embryo transfer procedures are:  recovering a large number of progeny from genetically superior females, easier introduction of exotic breeds, preserving endangered species, the opportunity for progeny testing females, minimizing the risk of introducing exotic diseases, minimizing cost and eliminating transportation stress in animals, obtaining twins and multiples from each pregnancy, utilizing genetically inferior females as foster mothers for embryos, and increasing the rates of genetic improvement (Ishwar and Memon, 1996; Earl and Kotaras, 1997; Gordon, 1997).

 

One of the technologies that increases the efficiency of embryo transfer is the in vitro production (IVP) of embryos.  This procedure permits the production of a large number of offspring from living or slaughtered animals (Gordon, 1997; Loi et al., 1998; Guler et al., 2000). However, in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) results in a reduced percentage of transferable embryos compared to in vivo conditions (Van Wagtendonk-de Leeuw et al., 1998). Therefore, numerous experiments have been performed to improve embryo IVP systems.

 

Numerous supplements including gonadotropins, steroid hormones, growth hormone and several growth factors including EGF have been added to maturation, fertilization and/or culture media in order to improve the efficiency of IVP systems (Watson et al., 1994; Longergan et al., 1996; Abeydeera et al., 2000; Guler et al., 2000).  Epidermal growth factor has been demonstrated to enhance oocyte maturation and blastocyst formation in several species (Coskun et al., 1991; Downs, 1989; Kim et al., 1999; Guler et al., 2000; Grazul-Bilska et al., 2001).  It has been indicated that EGF can also have an effect on implantation (Kim et al., 1999). 

 

The aim of this study was to evaluate the pregnancy rates after transfer of in vitro embryos produced in the presence or absence of EGF during in vitro maturation.

 

MATERIALS AND METHODS

 

Ewes of mixed breeds (n = 15) were injected twice daily (morning and evening) with FSH-P (Sioux Biochemical, Sioux Center, IA; Jablonka-Shariff et al., 1994; Stenbak et al., 2001) on days 13 (5 units/injection, day 0 = estrus) and 14 (4 units/injection) of the estrous cycle.  On the morning of day 15,

ovariectomy was performed (Reynolds et al., 1998). The number of visible follicles on each ovary was counted.  Oocytes were isolated by opening each visible follicle using a no. 15 scalpel blade and flushing it with oocyte collection medium two to three times using a Pasteur pipette in a petri dish (Watson et al., 1994; Stenbak et al, 2001; Grazul-Bilska et al., 2001.  By using a stereoscope, oocytes were recovered from each dish and transferred to a petri dish containing fresh collection medium without heparin. Oocytes were then evaluated and categorized as healthy or atretic based on morphology according to Thompson et al. (1995). All oocytes were washed three times in maturation medium (TCM-199 containing 10 % FBS, ovine FSH [oFSH-RP-1; NIAMDD-NIH, Bethesda, MD], ovine LH [oLH-26; NIADDK-NIH], estradiol [Sigma], glutamine [Sigma], sodium pyruvate [Sigma], and penicillin/streptomycin; Stenbak et al, 2001).  Before use, maturation medium was stabilized by incubating it overnight under oil at 39ºC in 5% CO2 and 95% air.  For each ewe, half of the oocytes were incubated in

 

maturation medium without EGF (control group), and the other half were incubated in the same medium containing 10 ng/ml EGF (Sigma; EGF group).  Dose of EGF was chosen on the basis of previously published data (Longergan et al., 1996; Abeydeera et al., 2000; Guler et al 2000; Grazul-Bilska et al., 2001).

 

The oocytes were matured for 24 h at 39ºC in 5% CO2 and 95% air followed by cumulus cells removal by using a 1% hyaluronidase (Type I-S; Sigma) treatment. The oocytes were again evaluated for health based on morphology (Thompson et al., 1995). Oocytes classified as healthy were used for IVF. The oocytes were transferred to stabilized fertilization medium, consisting of synthetic oviductal fluid (SOF; Tervit et al., 1992) and 2% heat inactivated sheep serum collected from sheep on day 0-1 of the estrous cycle (O’Brien et al., 1997; Brown and Radziewic, 1998; Wang et al., 1998).

 

Frozen semen, which was pooled from 4 Hampshire rams, was thawed and viable sperm were separated using the swim up technique (Yovich, 1995; Stenbak et al, 2001). In  this procedure,

healthy and viable sperm from a semen fraction swim into the medium (modified sperm washing medium: Irvine Scientific, Santa Ana, CA), which lies on top of the thawed semen pool. This media containing the motile sperm was then centrifuged, the sperm were counted and

 0.5-1.0x106 sperm/ml were added to the oocytes (up to 20 oocytes/500 µl/well).  The oocytes were incubated with the sperm in fertilization media for 18 h at 39ºC, 5% O2, 5% CO2 and 90% N2. The embryos were then washed three times with culture medium without glucose (SOF supplemented with BSA, glutamine, MEM amino acids, BME amino acids [Sigma] and penicillin/ streptomycin; Catt et al., 1997; Stenbak et al, 2001), and cultured in the same medium for 24 h at 39ºC, 5% O2, 5% CO2 and 90% N2. The dishes were then evaluated to determine the number of fertilized oocytes.  The fertilized oocytes were washed three times in culture medium containing glucose (Stenbak et al., 2001).  After 48 hours, the developmental stage of the zygotes was evaluated and embryos in the stage of 16 or more cells were randomly selected for transfer on day 5 (day1 = day of fertilization).

 

Thirty-nine ewes of mixed breeds were selected to receive embryos on day 5 of the estrous cycle. The estrous cycles of the recipient ewes had been synchronized so that their expected day of ovulation coincided with the day of IVF.  Synchronization consisted of a  single i.m. injection of PGF2a (Estrumate, Schering-Plough Animal Health Corp., Union, NJ. 1 cc, 0.5 doses) on day 8-12 of the estrous cycle.

 

For embryo transfer, recipient ewes were anesthetized as described by Gourley and Riese (1998).  The abdominal cavity was inflated with CO2 and a 1.5 mm diameter laparoscope was inserted through a 2 mm trocar approximately 5 cm anterior to the udder, and approximately 2 cm lateral to the midline.  The ovary containing the functional corpus luteum (CL) was identified.  A 5 cm incision was then made through the skin and body wall adjacent to the laparoscope and approximately 2-5 cm lateral to the midline. A Babcock forceps was inserted through this incision and the anterior portion (approximately 4-5 cm) of uterine horn ipsilateral to the ovary with the CL was clamped exteriorized.  A Sovereign̉ Tom Cat catheter (Sherwood Medical, St Louis, MO, USA) containing two or three embryos in about 3 mL of culture medium was inserted approximately 2 to 4 cm into the tip of the uterine lumen through a small incision and the embryos were injected.  The uterine horn was then replaced into the abdomen and the abdominal incision was sutured.

 

Before surgery, the recipients were on a pelleted diet as follows (% of diet dry matter): dehydrated beet pulp, 48.5%; dehydrated alfalfa, 24.3%; corn, 24.3%; soybean meal, 3.0%.  This was fed at a level (dry matter intake) of 862 g/d, which worked out on an as-fed basis to about 958 g/d assuming 90% dry matter. Immediately after surgery (day 0), the ewes received 0.5 kg of chopped grass hay plus 1/3 the normal amount (0.32 kg) of the pelleted diet. On the day after surgery (day 1), ewes were fed a 0.5 kg of chopped grass hay plus 2/3 the normal amount (0.63 kg) of the pelleted diet. On the second day after surgery (day 2), ewes received no grass hay but they received the full amount (1 kg) of the pelleted diet. On the third day after surgery (day 3), ewes were put back on the normal feeding schedule. To verify pregnancy, the recipient ewes were placed with vasectomized rams beginning on day 6 after embryo transfer.  In addition, the presence of fetuses was determined by ultrasonography (Classic Ultrasound Equipment Ltd, Tequesta, FL) on day 45 or after of embryo transfer.

 

All data are reported as means ± standard error (SEM). Data were analyzed by using the general linear models (GLM) procedure of the Statistical Analysis System (SAS, 1985) with the main effect of EGF. In addition, data for the percentage of oocytes cleaved and the percentage of pregnancy were analyzed by Chi-square (Spiegel, 1961).          

 

 

RESULTS

 

The number of follicles was 22.9 ± 1.4/ewe, number of recovered oocytes was 23.9 ± 1.7/ewe, number of healthy oocytes was 21.6 ± 1.6/ewe, number of atretic oocytes was 2.3 ± 0.7/ewe and percentage of healthy oocytes was 90.5 ± 1.9 %/ewe.  Total number of oocytes used for IVF was 370; 166 oocytes were matured with EGF, and 204 oocytes were matured without EGF.

 

EGF affected the morphology of the cumulus oocyte complex (COC).  After maturation, cumulus cells were more expanded in cultures with EGF than without EGF.  Table 1 presents the number of oocytes used for IVF and the number and percentage of fertilized oocytes after maturation with or without EGF.

 


Table 1. The number of oocytes used for IVF and number and percentage of fertilized oocytes after maturation with or without EGF.

 

Treatment

Group

No. of oocytes used for IVF

No. of fertilized oocytes (%)

No. of oocytes not fertilized (%)

Total

Per ewe

Total

Per ewe

Total

Per ewe

Control

204

12.8 ± 1

120*

(59%*)

7.5 ± 0.8

84

(35%)

5.3 ± 0.8

EGF

166

11.9 ± 1.1

129

(78%)

9.2 ± 0.9

37

(25%)

2.6 ± 0.8

* Values differ within a column (P<0.05)

 

The fertilization rate was greater (P<0.05) for oocytes matured with EGF (78%) than without EGF (59%).

 

Table 2 shows the effects of EGF on the rate of pregnancy.

 

Table 2.  Pregnancy rates after transfer of embryos produced by IVF after maturation with or without EGF.

 

Treatment Group

No. of recipient ewes

No. of pregnancies (%)

Control

18

7 (39)

EGF

21

11 (52)

 

Pregnancy rates were similar (P > 0.1) for both treatment groups.

 

DISCUSSION

 

Although it has been reported that epidermal growth factor plays an important role in the control of cell proliferation and differentiation in general, EGF involvement in the regulation of early embryonic development and implantation is poorly understood.

 

The data of the present experiment demonstrated that presence of EGF in maturation medium resulted in a 19% increase (78% vs. 59%) in the rate of fertilization but did not affect pregnancy rates after transfer of embryos produced in vitro.  Previous reports demonstrated that EGF did not affect the rates of fertilization but increased the rate of blastocyst formation in sheep (Guler et al., 2000; Grazul-Bilska et al., 2001). In the present experiment, the rate of blastocyst formation was not determined since embryos were transferred too early (on day 5 after fertilization) for blastocyst to be formed.  The discrepancies between studies in terms of the effects of EGF on the rates of fertilization are likely due to culture conditions, breed and condition of ewes.  However, these reports indicate that EGF in maturation medium is desirable to obtain a large number of in vitro produced embryos for further transfer.

 

Similar rates of fertilization of ovine oocytes, which were matured and fertilized in medium under various conditions but without EGF, were reported by others (60 % by Wang et al., 1998; 70 % by Stenbak et al., 2001; 72 % by Ledda et al., 1997; 82 % by O’Brien et al., 1997; 74 % by Watson et al., 1994).   This suggests that some specific culture conditions may provide a similar environment for high fertilization rates and further embryonic development as in our present experiment.

 

The pregnancy rates were similar for both groups (overall 46 %).  Pregnancy rates reported by others varied from 29 to 65 % (Rexroad and Powell, 1990; Slavik et al., 1992; Thompson et al., 1995; Brown et al., 1998; Ptak et al., 1999).  These differences may be due to the in vitro culture conditions, stage of development of transferred embryos, hormonal treatment, breed and location.  However, our experiment demonstrated that transfer of embryos on day 5 is very efficient.  Transfer of embryos on day 5 allows for shortening the time of embryo culture and the application of a laparoscopical technique for transferring embryos to the recipient ewes.

 

In summary, these data demonstrate that the presence of EGF in maturation medium increases the rate of fertilization but does not affect the rate of pregnancy in ewes during the normal breeding season.  In addition, transfer of embryos on day 5 resulted in a relatively high and satisfactory rate of pregnancy. 

 

ACKNOWLEDGMENTS

 

Supported in part by Hatch Project ND 01705 and ND SBARE.  The authors thank Dr. Joel Caton for expert technical assistance with feed method

after embryo transfer, and Wes Limesand for technical assistance in animal care.

 

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