Effects of overnutrition and undernutrition on in vitro fertilization (IVF) and early embryonic development in sheep

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1 Effets of overnutrition nd undernutrition on in vitro fertiliztion (IVF) nd erly emryoni development in sheep A.T. Grzul-Bilsk, E. Borowzyk, W. Arndt, J. Evoniuk, M. O Neil, J.J. Bilski, R.M. Weigl, Jmes D. Kirsh, Kim C. Krft, K.A. Vonnhme, D.A. Redmer, L.P. Reynolds nd J.S. Cton. Deprtment of Animl nd Rnge Sienes, North Dkot Stte University, Frgo, ND, USA ABSTRACT Nutrition hs een shown to influene severl reprodutive funtions inluding hormone prodution, ooyte ompetene nd fertiliztion, nd erly emryoni development. To determine effets of mternl diet on in vitro fertiliztion (IVF) nd erly emryoni development, ewes (n = 48; 58.4 ± 1.7 kg initil ody weight [BW]; 2.3 ± 0.1 initil ody ondition sore [BCS]) were divided into ontrol, overfed (d liitum feeding) nd underfed (60% of ontrol) nutritionl plnes for 8 weeks efore ooyte olletion. Ewes were individully fed one dily with pelleted diets ontining 2.4 Ml of ME/kg nd 13% CP (DM sis). Control ewes were fed to mintin BW nd offered 760 g/dy/50 kg. Synhroniztion of estrus ws performed using progestgen sponges for 14 dys. Folliulr development ws indued y twie dily injetions of follile stimulting hormone (FSH) on dys 13 nd 14 of the estrous yle. During the 8 week experiment, ontrol ewes lost 1.0 ± 0.9 kg, overfed ewes gined 11.8 ± 1.1 ut underfed ewes lost 14.2 ± 0.9 kg, nd BCS inresed y 0.7 ± 0.2 nd y 2.0 ± 0.1for ontrol nd overfed ewes, respetively, ut deresed y 0.7 ± 0.1 for underfed ewes to ompre with initil BCS. Ooytes were olleted from ll visile folliles on dy 15 of the estrous yle. After IVF, the proportion of developing emryos ws evluted throughout the 8 dy ulture period. Overnutrition nd undernutrition deresed (P < ) rtes of levge, nd morul nd lstoyst formtion (from 85% to 51% nd 48%; from 63% to 18 nd 28% nd from 40% to 5% nd 6% for ontrol, overfed nd underfed ewes, respetively). However, numer of visile folliles (lrge nd smll), totl numer of ooytes, numer of helthy ooytes nd perentge of helthy ooytes were similr for ontrol nd underfed ewes. These dt indite tht overnutrition nd undernutrition of donor ewes resulting in lower BW nd BCS, hs negtive effet on ooyte qulity whih results in lower rtes of levge, nd morul nd lstoyst formtion. These dt demonstrte tht nutrition level sustntilly ffet IVF nd erly emryoni development. Key words: overnutrition, undernutrition, ssisted reprodution, IVF, emryo, sheep INTRODUCTION Assisted reprodutive tehnologies (ART) hve mny pplitions in griulture. Reserh direted towrd improved qulity of ooytes nd in vitro emryo prodution hs predominntly foused on optimiztion of ulture onditions (Thompson, 1997; Guler et l., 2000; Rizos et l., 2002) nd/or diet mnipultion of donor ewes (O Cllghn et l., 2000; Lozno et l., 2003; Peur et l., 2003) nd ows (Yku et l., 1999; Sinlir et l., 2000; Armstrong et l., 2001). Nutritionl sttus is mjor ftor influening n niml s ility to reprodue (Roinson, 1990; We et l., 1999; O Cllghn et l., 2000). Nutrition hs signifint impt on

2 numerous reprodutive funtions inluding hormone prodution, fertiliztion nd erly emryoni development (Bolnd et l., 2001; Armstrong et l., 2003; Bolnd nd Lonergn, 2005). Nutritionl sttus hs een orrelted with emryo survivl nd is key ftor influening effiieny in ART (Armstrong et l., 2003; We et l., 2004). Confliting results hve een reported for the effets of low or high energy diets on ooyte qulity nd erly emryoni development in ruminnts (Kendrik et l., 1999; Bolnd et l., 2001; Ppdopoulos et l., 2001). For exmple in sheep, low energy diets deresed levge rtes ompred with high energy diets (Ppdopoulos et l., 2001). In ontrst, higher proportion of ov from ewes on low lorie diets were onsidered vile ompred with those from ewes on high lorie diets (MEvoy et l., 1995). For ows, positive (Noln et l., 1998; Kendrik et l., 1999; Bolnd et l., 2001), negtive (Yku et l., 1999; Armstrong et l., 2001) or no effets (Tripp et l., 2000) of plne of nutrition (high or low energy diets) on ooyte qulity, fertiliztion rte nd erly emryoni development hve een reported. Therefore, dditionl study should lrify the effets of nutritionl plne on ooyte qulity nd erly emryoni development. We hypothesized tht overfeeding or underfeeding of donor ewes would lter ooyte qulity s mesured y the rtes of fertiliztion nd erly emryoni development in vitro. Therefore, the im of the present study ws to evlute the effets of nutritionl plne (ontrol vs. overfed or underfed) on folliulr development, in vitro fertiliztion (IVF), nd erly emryoni development in FSH-treted ewes. Tretment of nimls MATERIALS AND METHODS Western rnge (predominntly Trghee nd Rmouillet) 2-3 yers old ewes were stndrdized for live weight nd ody ondition sore (BCS). Ewes were housed nd fed in individul pens (0.86 x 1.47 m) t the Animl Nutrition nd Physiology Center under 14 h of drkness nd 10 h of light t 12 C with free ess to wter nd minerl supplements. Ewes were divided into three groups: ontrol (n = 13 ewes) reeived mintenne diet (see elow), overfed (n = 18 ewes) fed d liitum, nd underfed (n = 17 ewes) reeived 60% of the mintenne diet for two months efore ooyte olletion. One week, during the durtion of the experiment ewes were weighed nd BCS ws evluted. Estrus ws synhronized y insertion of hrono-gest sponges (Intervet, UK) to the uterus for 14 dys. By using vsetomized rms, estrus ws deteted hours fter sponge withdrwl. Ewes reeived twie dily (morning nd evening) injetions with FSH-P (Sioux Biohemil, Sioux Center, IA, USA) on dys 13 (5 mg/injetion) nd 14 (4 mg/injetion) following estrus (dy 0) s desried efore (Stenk et l., 2001). On dy 15 of the estrus yle ewes were ovrietomized (Luther et l. 2005). The study ws initited during the norml reeding seson in August nd finished in Novemer. All proedures were performed t the niml experimentl filities of North Dkot Stte University (NDSU) loted in Frgo, ND, USA (pproximtely 46.9 ltitude nd longitude) nd were pproved y the Institutionl Animl Cre nd Use Committee of NDSU. Nutritionl mngement After rrivl nd 3 dy dpttion to individul pens nd pelleted diets, ewes were lloted rndomly to three nutritionl groups s desried ove. The diet ontined:

3 dehydrted eet pulp, 36.5%; dehydrted lflf, 20.3%; orn, 24.2%; soy hulls, 16%; soyen mel, 3.0% (% of dietry DM). The pelleted (0.48 m dimeter) diet, whih ws prepred nd nlyzed on site, supplied 2.4 Ml/ME nd 130 g rude protein (13%) per kg of diet DM sis nd ws offered in one portion dily. Dietry mngement proedures for oth groups were similr to those desried y Sheffer et l., 2004,. The mintenne diet, whih ws prepred nd nlyzed on site on weekly sis, supplies 2.4 Ml/kg of metolizle energy nd 130 g rude protein (13%) per kilogrm diet nd ws offered in one equl rtion dily. The ontrol group reeived 760 g/dy/50 kg (100%), overfed group ws fed d liitum (200% or more of ontrol) nd the underfed group reeived 456 g/dy/50 kg (60%) of the mintenne diet (dry mtter sis). Ooyte olletion Following ovrietomy, ovries were immersed in PBS nd trnsported to the lortory in n inutor t 39 C. The numer of visile smll (< 3mm) nd lrge (> 3mm) folliles on eh ovry ws determined, nd umulus ooyte omplexes (COC) were isolted y opening eh visile follile with slpel lde nd flushing it two to three times with ooyte olletion medium (Grzul-Bilsk et l., 2003, 2005; Luther et l., 2005). Under stereomirosope, COC were reovered from eh dish nd trnsferred to petri dish ontining fresh olletion medium without heprin. Cumulus ooyte omplexes were then evluted nd tegorized s helthy or treti sed on their morphology (Thompson et l., 1995). All COC were then wshed three times in mturtion medium (TCM-199 ontining 10% fetl ovine serum, ovine FSH [5 µg/ml; ofsh-rp-1; NIAMDD-NIH, Bethesd, MD, USA], ovine LH [5 µg/ml; olh-26; NIADDK-NIH], estrdiol -17β [1 µg/ml; Sigm St. Louis, MO, USA], glutmine [2 mm; Sigm], sodium pyruvte [0.25 mm; Sigm], epiderml growth ftor [10 ng/ml; Sigm,] nd peniillin/streptomyin [100 units/ml peniillin nd 100 µg/ml streptomyin; Gio, Grnd Islnd, NY, USA]; Grzul-Bilsk et l. 2003, 2005; Luther et l. 2005). Totl numer of ooytes used for IVF ws 162 for ontrol, 264 for overfed nd 232 for underfed ewes. In vitro mturtion Ooytes were mtured in vitro in mturtion medium for 24 h t 39 C in 5% CO 2 nd 95% ir followed y umulus ell removl using 1% (wt/vol) hyluronidse (Type I; Sigm) in PBS. The ooytes were gin evluted for helth sed on morphology. Ooytes lssified s helthy were used for IVF nd were trnsferred to equilirted fertiliztion medium onsisting of syntheti ovidutl fluid (SOF) prepred in our lortory (Stenk et l., 2001) nd 2% hetintivted sheep serum olleted from sheep on dy 0-1 of the estrous yle (Grzul-Bilsk et l., 2003, 2005; Luther et l., 2005). In vitro fertiliztion nd emryo ulture Frozen pitted semen pooled from 4 Hmpshire rms ws thwed nd vile sperm were seprted using the swim up tehnique (Grzul-Bilsk et l., 2005). The sperm (0.5 to 1.0 x 10 6 sperm/ml) were dded to the IVF medium ontining ooytes nd inuted for 18 h t 39ºC, 5% O 2, 5% CO 2 nd 90% N 2. The presumptive zygotes were then wshed three times with ulture medium without gluose (SOF supplemented with BSA, glutmine, MEM non-essentil

4 mino ids, BME mino ids [Sigm] nd peniillin/streptomyin) nd ultured in the sme medium for 24 h t 39ºC, 5% O 2, 5% CO 2 nd 90% N 2 (Grzul-Bilsk et l., 2003, 2005). The dishes were then evluted to determine the numer of leved ooytes. The emryos were trnsferred to ulture medium ontining gluose (1.5 mm). After 48 h, the developmentl stge ws evluted nd emryos were trnsferred to fresh ulture medium with gluose. The rte of levge (numer of leved vs. non-leved ooytes), nd the rte of erly emryoni development (time nd perentge rehing stge of morul or lstoyst) were evluted every seond dy during 8 dy ulture. Sttistil nlysis To ompre hnges in BW, verge dily gin (ADG) nd BCS during the experiment, numer of folliles nd ooytes, nd ooyte qulity vriles (e.g., the rtes of levge, nd morul nd lstoyst formtion) for ontrol nd underfed ewes, dt were nlyzed sttistilly y using the GLM progrm of SAS (SAS Inst., In., Cry, NC). Mens were seprted using the method of lest signifint differene. In ddition, dt for the proportion of overfed nd underfed ewes providing ooytes whih developed to lstoyst stge were nlyzed y Chisqure. RESULTS At the time tretment ws initited, BW ws similr for ontrol, overfed nd underfed groups (56.7± 2.1, 60.7± 1.2 nd 57.8 ± 1.8 kg respetively; Figure 1A). At the end of 8 week the experiment, BW of overfed ewes ws greter (P<0.01) thn ontrol or underfed ewes, nd BW of underfed ewes ws lower (P < 0.05) thn ontrol ewes (Figure 1A). Chnges in BW were signifintly different (P < 0.001) for nutrition groups t week 8 (Tle 1). Averge dily gins were greter or lower (P < 0.001) for overfed or underfed ompred with ontrol ewes during the during 8 week of experiment, respetively (Tle 1). When ompred to initil BW, ontrol ewes lost 1.0 ± 0.9 kg, overfed ewes gined 11.8 ± 1.1 ut underfed ewes lost 14.2 ± 0.9 kg over the 8 week experiment. At the eginning of tretment, BCS ws similr for ontrol, overfed nd underfed ewes (2.3 ± 0.1, 2.3 ± 0.1 nd 2.4 ± 0.1 respetively; Figure 1B). Body ondition sore of overfed ewes ws greter (P < 0.001) thn ontrol or underfed ewes t week 8 of the experiment (Figure 2B). Chnges in BCS were signifintly different (P < 0.001) for nutrition groups t week 8 (Tle 1). During 8 week experiment, BCS inresed y 0.7 ± 0.2 nd y 2.0 ± 0.1 for ontrol nd overfed ewes, respetively, ut deresed y 0.7 ± 0.1 for underfed ewes to ompre with initil BCS. Men numer of visile folliles, numer of lrge nd smll folliles, totl numer of ooytes, numer of helthy ooytes, perentge of helthy ooytes, nd the numer of ooytes used for IVF per ewe were similr for ontrol, overfed nd underfed groups (Tle 1). The numer of ooytes leved, levge rtes, numer of morul nd lstoysts nd the rtes of morul nd lstoyst formtion were greter (P < to 0.057) for ontrol ompred with overfed or underfed ewes (Tle 1). Proportion of ewes providing ooytes whih developed to lstoyst stge tended (P<0.1) to e greter in underfed thn overfed ewes.

5 Tle 1. Effets of nutrition on the folliulr development, the numer olleted ooytes, the numer of helthy ooytes, the rtes of levge nd morul nd lstoyst formtion, nd BW, ADG nd BCS Prmeter Control Overfed Underfed P vlue Numer of ewes Totl folliles (n) 27.5 ± ± ± Lrge folliles (n) 12.9 ± ± ± Smll folliles (n) 14.5 ± ± ± Totl Ooytes (n) 26.1 ± ± ± Helthy ooytes (n) 22.5 ± ± ± Helthy ooytes (%) 86.1 ± ± ± Ooytes used for IVF 12.5 ± ± ± Cleved ooytes (n) 10.5 ± ± ± Clevge rte (%) 84.8 ± ± ± Morul (n) 6.5 ± ± ± Morul (%) 63.2 ± ± ± Blstoyst (n) 4.2 ± ± ± Blstoyst (%) 40.1 ± ± ± Initil BW (kg) 56.7 ± ± ± Finl BW (kg) 55.7 ± ± ± Differene in BW (kg) -1.0 ± ± ± ADG (kg) ± ± ± Initil BCS 2.3 ± ± ± Finl BCS 3.0 ± ± ± Differene in BCS 0.7 ± ± ± *All vlues (men ± SEM) re expressed per ewe.

6 80 70 A BW (kg) Control Overfed Underfed 30 week 0 week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 5 B 4 BSC 3 Control Overfed Underfed 2 1 week 0 week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 Figure 1. Body weight (A) nd BCS (B) in ontrol (lk rs), overfed (grey rs) nd underfed (open rs).,, P < ; Mens ± SEM with different supersripts differ within speifi week.

7 DISCUSSION The present study demonstrted tht overfeeding nd underfeeding of ewes resulted in lower BW nd BCS when ompred to ontrol ewes. Furthermore, ooytes derived from overfed nd underfed ewes yielded fewer zygotes, moruls nd lstoysts nd hd lower rtes of levge, nd morul nd lstoyst formtion ompred with ooytes derived from ontrol ewes. In previous studies, for mture ewes fed low energy diet (pproximtely times mintenne energy requirement) for 3 to 4 week, deresed BCS (from 2.61 to 2.1 nd from 2.5 to 2.33; Aei et l., 1999 nd Lozno et l., 2003, respetively) ws oserved long with deresed levge rtes, numer of good qulity emryos nd the rtes of pregnny. On the other hnd, ewes d liitum feeding for pproximtely 3 week resulted in enhned BCS (from 2.58 to 2.7), ut lower superovultion responses, lower numer of good qulity ooytes nd emryos, nd greter perentge of poorly developed emryos in mture ewes (Lozno et l., 2003). In ddition, Snijders et l. (2000) demonstrted tht rtes of levge nd lstoyst formtion from ooytes derived from ows with BCS were lower (70.4 vs. 77.4%, nd 6.8% vs. 11.4%, respetively) ompred with those from ows with BCS during the first or third lttion. These dt nd lso our dt indite tht deresed or inresed BCS my e ssoited with deresed ooyte qulity mesured y the rtes of in vitro fertiliztion nd erly emryoni development in sheep. This suggests tht BCS n e used to predit suessful emryoni development in sheep. However, determintion of BCS is reltively sujetive nd my vry from study to study. In the present experiment, nutritionl plne hd no effet on the numer of ovrin folliles. Peur et l. (2003) reported for dult ewes tht low (0.7 x) or high (1.3 x) mintenne diets for 3 to 5 months efore FSH-indued superovultion did not ffet ovultion rtes. Moreover, superovultory responses fter FSH-tretment were not ffeted y 0.5 x, 1.0 x or 1.5 x mintenne diet fed during peri-oneption period in dult ewes (Kkr et l., 2005). These dt indites tht these speifi nutritionl tretments did not ffet folliulr development mesured y the numer of folliles or ovultions in sheep. The numer of folliles ws similr in ltting diry ows (not treted with FSH) reeiving low (1.52 Ml NE l /kg DM) or high (1.78 Ml NE l /kg DM) energy diet for pproximtely 25 week postprtum (Kendrik et l., 1999) nd in yerling eef heifers (not treted with FSH) fed d liitum or 0.75 x d liitum for 100 d (Tripp et l., 2000). On the other hnd, FSH-treted eef heifers fed low energy (9.6 Ml/kg ME/d) diet for 17 to19 dys hd more folliles thn ows fed high energy (28.6 Ml/kg ME/d) diet (Noln et l., 1998). Thus, these results show tht nutritionl tretments hd no effet on the numer of visile folliles in sheep ut not in ows. Moreover, numer of folliles in the present study ws similr to tht previously reported for FSH-treted mture ewes fed mintenne diet during the norml reeding seson nd sesonl nestrus (Stenk et l., 2001; Grzul-Bilsk et l., 2003; Luther et l., 2005). The levge rtes were lower for overfed (51%) or underfed (48%) ompred with ontrol (85%) ewes in our study. Ppdopoulos et l. (2001) hve lso shown tht levge rtes were deresed (from 88% to 66%) in ewes fed low energy (0.5 x mintenne energy requirements) diet in omprison with high energy (2 x mintenne energy requirements) diet for 28 dys. Similr to our results for overfed or underfed ewes, low levge rtes, 51% nd 35%, were oserved for ewes underfed (0.5 x mintenne energy requirements) nd overfed (d liitum intke) for pproximtely 24 dys, respetively (Lozno et l., 2003). This indites tht

8 indequte diet (e.g., underfeeding or feeding d liitum) ffets ooyte qulity mesured y IVF rtes in sheep. In ddition, rtes of levge similr to levge rtes in our ontrol group were reported for mture sheep fed mintenne diet (Wtson et l., 1994; Ledd et l., 1997; O Brien et l., 1997). Thus, the rtes of fertiliztion my e influened y different nutritionl regimens under whih ooytes were developed in the mternl environment. In the present study, the numer of moruls nd lstoysts nd the rte of morul nd lstoyst formtion were lower for overfed nd underfed ewes ompred with ontrol ewes. In ontrst, Lozno et l. (2003) demonstrted tht restrited diets (0.5 x of mintenne diet for out 24 dys) did not ffet the rtes of lstoyst formtion in mture sheep. Moreover, supplementtion with ure to the diet with low energy (0.5 x of mintenne) did not ffet the lstoyst ell numer nd lstoyst hthing rte in sheep (Ppdopoulos et l., 2001). Studies of MEvoy (1995) demonstrted tht emryos in the erly stge of development, produed in vivo nd then ultured in vitro olleted from mture ewes fed low lories (0.6 x mintenne) diet for out 2 week, were onsidered more vile, hd greter protein synthesis index nd numer of nulei in the lstoyst ompred with those produed in ewes fed high lorie diet. In ddition, the greter numer of ells in lstoysts produed in vivo ws oserved for ewes fed low lories diet (0.5 x mintenne diet) to ompre with ewes fed 1 x or 1.5 x mintenne diet during peri-oneption period (Kkr et l., 2005). Dt from these two studies indite tht ewe is le to respond to ute hnges in nutrition during peri-oneption period, resulting in hnges of emryoni development. For yerling eef heifers, restrition of dietry energy (75% of d liitum fed) did not ffet the rte of lstoyst formtion (Tripp et l., 2000). Therefore, it seems tht the level of overfeeding or feed restrition nd/or length of speifi feeding reported in some studies were not severe enough to indue negtive impt on the rte of lstoyst formtion, whih ws oserved in the urrent study. Moreover, the effets of nutritionl tretment on lstoyst formtion my lso depend on speifi diet omposition nd reed. Numerous experiments indite tht nutrition hs diret effets on some reprodutive funtion y ffeting hormonl prodution (O Cllghn nd Bolnd, 1999; Luy, 2003; Hunter et l., 2004). For exmple, for underfed or overfed mture ewes with enhned or deresed lood progesterone onentrtions, respetively, ltered ooyte nd emryo qulity ws oserved (Lozno et l., 2003). This indites tht effets of nutrition on ooyte nd emryoni development my e indiretly linked through regultion of hormone seretion. In the present study, we hve not evluted the level of hormones in peripherl lood. Therefore, future studies should e undertken to further define ssoition etween hormone levels nd ooyte qulity. The effets of nutrition on ooyte nd emryoni development my reflet the generl energy lne (e.g., mintenne diet vs. low or high energy diets) ut lso n e ttriuted to the speifi nutrients in diets, suh s vitmins, minerls nd other supplements (Wrenzyki et l., 2000). For exmple, Trin et l. (1998) oserved tht supplementtion with mixture of vitmins C nd E to the mternl diet enhned the numer of ovultions ut did not ffet the rtes of levge or lstoyst formtion in mie. Moreover, MEvoy et l. (1997) demonstrted tht high onentrtion of ure in diet fed for 12 week inresed emryo mortlity nd deresed pregnny rtes fter emryo trnsfer in mture sheep. Additionl studies should e undertken to determine whih nutritionl ftors ffet ooyte qulity. In onlusion, our results demonstrte tht overnutrition nd undernutrition did not ffet numer of developing folliles, numer of reovered ooytes, numer nd perentge of helthy ooytes per ewe, ut deresed numer of leved ooytes, nd the rtes of fertiliztion nd

9 morul nd lstoyst formtion. These dt indite tht donor nimls likely require speifi nutritionl mngement proedures to provide the highest qulity ooytes for ART. Nutrition of donor nimls seems to e key omponent ffeting development of ooytes nd the preimplnttion emryo. We mnipulted totl dietry intke in the present study, ut future investigtions tht ddress speifi dietry nutrient omposition should provide insight into the underlying mehnisms ssoited with hnges in effiieny of in vitro emryo prodution. ACKNOWLEDGEMENTS The uthors would like to thnk Mr. Tim Johnson, Mr. Wes Limesnd nd Mr. Terry Skunerg, nd other memers of our lortory for their tehnil ssistne. LITERATURE CITED Aei, J. A., F. Ford, nd J. M. Lozno A preliminry report on the effet of dietry energy on prostglndin F2lph prodution in vitro, interferon-tu synthesis y the oneptus, endometril progesterone onentrtion on dys 9 nd 15 of pregnny nd ssoited rtes of emryo wstge in ewes. Theriogenology 52: Armstrong, D. G., J. G. Gong, nd R. We Intertions etween nutrition nd ovrin tivity in ttle: physiologil, ellulr nd moleulr mehnisms. Reprodution Suppl. 61: Armstrong, D. G., T. G. MEvoy, G. Bxter, J. J. Roinson, C. O. Hogg, K. J. Wod, R. We, nd K. D. Sinlir Effet of dietry energy nd protein on ovine folliulr dynmis nd emryo prodution in vitro: ssoitions with the ovrin insulin-like growth ftor system. Biol. Reprod. 64: Bolnd, M. P., P. Lonergn, nd D. O'Cllghn Effet of nutrition on endorine prmeters, ovrin physiology, nd ooyte nd emryo development. Theriogenology 55: Bolnd, M. P. nd P. Lonergn Effets of nutrition on fertility in diry ows. Adv. Diry Tehn. 15: Grzul-Bilsk, A. T., J. T. Choi, J. J. Bilski, R. M. Weigl, J. D. Kirsh, K. C. Krft, L. P. Reynolds, nd D. A. Redmer Effets of epiderml growth ftor on erly emryoni development fter in vitro fertiliztion of ooytes olleted from ewes treted with follile stimulting hormone. Theriogenology 59: Grzul-Bilsk, A. T., D. Pnt, J. S. Luther, P. P. Borowiz, C. Nvnukrw, J. S. Cton, M. A. Wrd, D. A. Redmer nd L. P. Reynolds Pregnny rtes nd grvid uterine prmeters in nturlly red ewes nd ewes fter trnsfer of in vitro produed emryos, nd in single, twin nd triplet pregnnies. Anim. Reprod. Si. (in press). Guler, A., N. Poulin, P. Mermillod, M. Terqui, nd Y. Cognie Effets of growth ftors, EGF nd IGF-1, nd estrdiol on in vitro mturtion of sheep ooytes. Theriogenology 54: Hunter, M.G., R.S. Roinson, G.E. Mnn, nd R. We Endorine nd prrine ontrol of folliulr development nd ovultion rte in frm speies. Anim. Reprod. Si : Kkr, M.A., S. Mddoks, M.F. Lorimer, D.O. Kleemnn, S.R. Rudiger, K.M.Hrtwih, nd S.K. Wlker The effet of peri-oneption nutrition on emryo qulity in the superovulted ewe. Theriogenology 64:

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