2010, Sheep & Goat Research Journal Sheep & Goat Research Journal, Volume 25, 2010 21

Summary Ewes were assigned to the following treatments to deter-

mine whether estradiol-17-oxytocin treatment affects lutealfunction and pregnancy rates on d 25: 1) diluent + saline (n =26); 2) diluent + oxytocin (n = 25); 3) estradiol-17 + saline(n = 22); and 4) estradiol-17 + oxytocin (n = 24). On d 6 afterexpected estrus and mating, ewes received either i.v. injectionof diluent or i.v. injection of 100 g of estradiol-17. Ten hourslater, ewes received either i.v. injection of saline or i.v. injectionof 400 USP units of oxytocin. Blood samples for progesteroneassay were collected on d 6, d 7, d 8 (Period 1), d 16, d 18, d 20,d 22, and d 25 (Period 2). Transrectal ultrasonography on d 25and progesterone concentrations were used to diagnose preg-nancy. Neither estradiol-17 nor oxytocin affected pregnancy

rates, and the estradiol-17 oxytocin interaction was not sig-nificant. The pregnancy rate for diluent + saline was 61.5 per-cent; diluent + oxytocin, 76 percent; estradiol-17 + saline,77.2 percent; and estradiol-17 + oxytocin, 62.5 percent. Prog-esterone concentration was greater (P < 0.05) in pregnant thanin nonpregnant ewes (5.2 0.3 ng/mL vs. 2.0 0.6 ng/mL); thepregnancy status period interaction was significant (P 0.05). Treatment with estradiol-17 on d 6after the expected onset of estrus and oxytocin 10 h later didnot induce luteolysis or disrupt pregnancy in ewes.

Key Words: Sheep; Embryo Transfer; Estradiol; Oxytocin;Transcervical

Pregnancy rates after ewes were treated withestradiol-17 and oxytocin

G. S. Lewis1

1 USDA, Agricultural Research Service, U.S. Sheep Experiment Station, 19 Office Loop, Dubois, Idaho 83423-5032

All correspondence to: Dr. Gregory S. Lewis, Research Leader; USDA, ARS, U.S. Sheep Experiment Station;

19 Office Loop; Dubois, Idaho 83423-5032; Phone: (208) 374-5306; Fax:(208) 374-5582; E-mail: Gregory.Lewis@ars.usda.gov

The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or

the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.

Volume 25, 2010

IntroductionSurgical procedures have been used

for at least 55 years to collect and trans-fer sheep embryos (Hunter et al., 1955;McKelvey et al., 1986; Nellenschulteand Niemann, 1992; Li et al., 2008).Sheep cervical anatomy makes nonsurgi-cal embryo collection and transferexceedingly difficult and considerablyless effective than surgical procedures(McKelvey et al., 1986; Flohr et al.,1999; Wulster-Radcliffe et al., 1999;Anel et al., 2006; Candappa et al.,2009). Even though some of the surgicalembryo transfer (ET) procedures areminimally invasive, they include anal-gesics, anesthetics, incisions, andsutures, and they require specializedtraining to perform.

Effective transcervical AI, embryocollection, and ET procedures for sheepmay eliminate the need for surgical pro-cedures. Sheep-specific transcervical pro-cedures have been reported, but preg-nancy rates after the procedures havebeen disappointing (Sayre and Lewis,1997; Wulster-Radcliffe et al., 1999,2004; Anel et al., 2006). Some of theprocedures have included treatments todilate the cervix and reduce the difficultyof manipulating a catheter through thecervix and into the uterus (Flohr et al.,1999; Wulster-Radcliffe et al., 2004;Anel et al., 2006; Candappa et al., 2009).

In one study, an i.v. injection of 100g of estradiol-17 on d 5 of an estrouscycle and an i.v. injection of 400 USPunits of oxytocin 10 h later were used todilate the cervix and permit atraumatictranscervical ET (Wulster-Radcliffe etal., 1999). Without the estradiol-17-oxytocin treatment, the atraumatic tran-scervical ET procedure described inWulster-Radcliffe et al. (1999) wouldnot have been possible. Based on post-mortem evaluations on d 14, the estra-diol-17-oxytocin treatment did notseem to reduce embryonic development,but the effects of the estradiol-17-oxy-tocin treatment per se on later preg-nancy rates were not determined (Wul-ster-Radcliffe et al., 1999). Thus, thisexperiment was conducted to determinewhether treating ewes with estradiol-17and oxytocin on d 6 and d 7, respec-tively, after the expected day of onset ofestrus and mating would affect preg-nancy rates on d 25.

Materials and Methods

Animals and Treatments

Estrus was synchronized using estab-lished procedures (Wulster-Radcliffe etal., 1999, 2004) during the autumnbreeding season in a group of crossbredewes (n = 97), that contained variouscombinations of white-faced and black-faced genetics. Ewes were mated natu-rally with rams immediately afterprogestogen withdrawal and i.m. injec-tion of 400 IU of eCG (Sioux Biochem-ical, Sioux City, Iowa). Ewes were notchecked for signs of estrus during anypart of the experiment.

Treatments were in a 2 2 factorialarray. Ewes were assigned to one of fourrandomized treatments: 1) diluent +saline (n = 26); 2) diluent + oxytocin (n= 25); 3) estradiol-17 + saline (n = 22);and 4) estradiol-17 + oxytocin (n =24). During the afternoon of d 6 after theexpected onset of estrus and mating (i.e.,48 h after progestogen withdrawal; Clineet al., 2001), approximately half of theewes received a 5-mL i.v. injection ofdiluent (50 percent ethanol:50 percentsterile, isotonic saline), and the remain-ing ewes received an i.v. injection of 100g of estradiol-17 (Sigma ChemicalCo., St. Louis, Mo.) in 5 mL of diluent.At 10 h after the initial injections (i.e.,morning of d 7), approximately half ofthe diluent-treated and half of the estra-diol-17-treated ewes received 20-mLi.v. injections of sterile, isotonic saline,and the remaining diluent-treated andestradiol-17-treated ewes received i.v.injections of 400 USP units of oxytocin(20 mL; Vedco, St. Joseph, Mo.; Khalifaet al., 1992; Wulster-Radcliffe et al.,1999). Injections were via a jugular vein.Estradiol-17 and oxytocin were admin-istered on d 6 and d 7, respectively,because sheep embryos are commonlytransferred during this portion of theestrous cycle (Li et al., 2008).

Blood Sampling

On d 6, d 7, d 8, d 16, d 18, d 20, d22, and d 25 after the expected onset ofestrus and mating, heparinized Vacu-tainer tubes (Becton Dickinson,Franklin Lakes, N.J.) were used to col-lect approximately 10 mL of jugularvenous blood from each ewe. Blood sam-ples were placed in an ice bath immedi-ately after collection and stored there

until approximately 1 h after the end ofeach collection period. Plasma was thenharvested after centrifugation and storedin polypropylene tubes at -20C.

Blood samples were collected justbefore injections were administered on d6 and d 7. Blood samples were not col-lected between d 9 and d 15 becauseprogesterone concentrations wereexpected to be similar between pregnantand nonpregnant ewes during those days(deNicolo et al., 2009).

Pregnancy Diagnosis

Transrectal ultrasonography 25 dafter the expected onset of estrus andprogesterone concentrations was used todiagnose pregnancy. An Aloka 500 Vinstrument equipped with a 5.0 MHz lin-ear array transducer (Corometrics Med-ical Systems, Inc., Wallingford, Conn.)was used. Ewes were restrained in dorsalrecumbency in a Poldenvale Com-modore cradle (Premier Sheep Supplies,Washington, Iowa) during the ultra-sound procedure. A ewe was consideredpregnant if 1) an embryo was visible inan ultrasound image, 2) a heartbeat couldbe visualized, and 3) progesterone con-centrations on d 16 through d 25 weregreater than 2.0 ng/mL. A heartbeatcould be detected in all embryos thatwere visualized. A ewe was considerednonpregnant if 1) an embryo was not vis-ible in an ultrasound image and 2) prog-esterone concentrations on d 16 throughd 25 were less than 2.0 ng/mL. Lambingdata could not be collected, as the ewesin this study were scheduled to be sold asexcess. However, the decision to sell aewe was not based on the lack of repro-ductive soundness. In addition, approxi-mately 20 percent of the ewes can beexpected to lose embryos or fetuses afterd 25, and these losses could compromisethe ability to determine the short-termeffects of the estradiol-17 and oxytocintreatments (Dixon et al., 2007).

Progesterone Assay

A solid-phase RIA [125I] progesterone(Diagnostic Products, Los Angeles, Calif.)was used to quantify progesterone in allplasma samples (Seals et al., 2002; Lewis,2003; Wulster-Radcliffe et al., 2003; Lewisand Wulster-Radcliffe, 2006).

Statistical Methods

Procedures for analyzing categorical

22 Sheep & Goat Research Journal, Volume 25, 2010 2010, Sheep & Goat Research Journal

2010, Sheep & Goat Research Journal Sheep & Goat Research Journal, Volume 25, 2010 23

data were used to determine the effectsof treatment on pregnancy rates, andgeneral linear-model procedures wereused to determine the effects of treat-ment on progesterone concentrations(SAS, Cary, N.C.). The model used todetermine the effect of treatment onpregnancy rate included terms for estra-diol-17, oxytocin, and the estradiol-17 oxytocin interaction.

The initial model used to analyzethe progesterone data included terms forestradiol-17, oxytocin, pregnancy sta-tus (i.e., pregnant or nonpregnant),period (i.e., Period 1 = d 6, d 7, and d 8;Period 2 = d 16, d 18, d 20, d 22, and d25), all interactions, and error terms.Data were then sorted according to preg-nancy status, and similar models wereused to determine the effects of treat-ment and period on progesterone con-centrations in pregnant ewes and non-pregnant ewes.

Results and DiscussionThe results of this experiment indi-

cate that neither estradiol-17 nor oxy-tocin administered on d 6 and d 7,respectively, after the expected onset ofestrus and mating affected pregnancyrates on d 25, and the estradiol-17 oxytocin interaction was not significant.The overall pregnancy rate was 69.1 per-cent (67/97), which was similar to the d25-pregnancy rates in Dixon et al.(2007). The pregnancy rate for diluent +saline was 61.5 percent (16/26); diluent+ oxytocin, 76 percent (19/25); estra-diol-17 + saline, 77.2 percent (17/22);and estradiol-17 + oxytocin, 62.5 per-cent (15/24). Embryos that were trans-ferred after estradiol-17-oxytocin-induced cervical dilation on d 6 of theestrous cycle appeared to survive anddevelop until they were evaluated post-mortem on d 14 of pregnancy (Wulster-Radcliffe et al., 1999). However, experi-ments must be conducted to determinewhether embryos that are transferredtranscervically after estradiol-17 andoxytocin treatment will survive to term.

Pregnancy status affected (P < 0.05)progesterone concentrations (pregnant,5.2 0.3 ng/mL of plasma vs. nonpreg-nant, 2.0 0.6 ng/mL of plasma), andthe pregnancy status period interac-tion was significant (P < 0.01; Table 1).However, estradiol-17 treatment, oxy-tocin treatment, and the estradiol-17

oxytocin interaction were not significant(P > 0.05; Table 1). In pregnant ewes,the concentration of progesterone dur-ing Period 1 (4.9 0.3 ng/mL) was less(P < 0.01) than it was during Period 2(5.4 0.4 ng/mL). In nonpregnant ewes,progesterone concentrations were less (P< 0.01) during Period 2 than they werefor all ewes during Period 1 and for preg-nant ewes during Period 2. Progesteroneconcentrations in nonpregnant ewes haddecreased (P < 0.05) to less than 1.0ng/mL by d 16, and they remained lessthan 1.0 ng/mL through d 25. The dif-ferences in progesterone concentrationsin pregnant and nonpregnant ewes andreduced progesterone concentrations innonpregnant ewes on d 16 through d 25after the expected onset of estrus andmating, compared with d 6 through d 8,are consistent with data from numerousother experiments (Lewis et al., 1981;Wulster-Radcliffe et al., 1999; deNicoloet al., 2009).

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