NORGESTOMET IMPLANTS ENHANCE EMBRYO SURVIVAL IN POSTPARTUM COWS: A PRELIMINARY REPORT

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1 NORGESTOMET IMPLANTS ENHANCE EMBRYO SURVIVAL IN POSTPARTUM COWS: A PRELIMINARY REPORT M. L. Rosmarin, T. F. Lock, J. M. Dahlquist, T. G. Nash, D. B. Faulkner, and D. J. Kesler SUMMARY Objectives of this study were to determine if norgestomet administered on days 5 to 24 post-ai would 1) facilitate embryo survival in cows with short luteal phases (< 10 days) and cows with premature corpus luteum regression (16-20 d post-ai) and 2) enhance the establishment of pregnancy. Beef cows suckling calves from the University of Illinois (n=138) were administered Syncro-Mate B (SMB) and bred via AI 48 hours after implant removal (48-h AI). Cows were 12 to 106 days postpartum at the 48-h AI and randomly assigned to one of two groups. Treated cows (n=69) were implanted with two norgestomet/silicone implants on the convex surface of the ear. The other 69 cows were not treated on days 5 to 24 post-ai (controls). Two implants released 315 µg norgestomet per day (> two times the dose that suppresses estrus and a dose that maintains pregnancy in ovariectomized heifers). Blood samples were collected before SMB implantation (to determine anestrus/estrus-cycling status) and twice weekly after SMB implant removal for 38 days and assayed for progesterone (P 4 ). All cows were observed twice daily for 31 days after SMB implant removal for estrus and bred via AI. Cows were examined for pregnancy via transrectal ultrasonography 24 and 38 days after AI. Qualitative data were analyzed by chi-square analysis. Pregnancy rate and P 4 were analyzed by split-plot analysis of variance. Norgestomet implants increased (P <.01) the 24 d pregnancy rate (pregnancy rates = 30% and 57% for control and norgestomet treated cows, respectively). The 24 d pregnancy rate (54%) for the previously anestrus cows administered norgestomet was not different (P >.20) from the untreated estrus-cycling cows. The 24 d pregnancy rate for the norgestomet treated cows with progesterone concentrations indicative of cows with subnormal luteal phases (60%) was not different (P >.20) from the pregnancy rate of untreated estrus-cycling cows (41%). The pregnancy rate decreased (P <.05) from d 24 to 38 in the norgestomet treated cows; however, the pregnancy rate on d 38 for the previously anestrus cows administered norgestomet was greater (P <.05) than for the untreated previously anestrous cows. Although norgestomet implants maintained pregnancy in the absence of corpora lutea, embryonic loss occurred after norgestomet implant removal on d 24 in the absence of corpora lutea. In summary, norgestomet enhanced embryo survival in cows with premature corpus luteum regression and cows with short luteal phases and may be incorporated into procedures to enhance the establishment of pregnancy. Also, cows with less than one standard deviation of mean P 4 concentrations of pregnant cows on days 6 or 7 would be poor recipient candidates for embryo transfer.

2 INTRODUCTION Calving rates of cattle bred after estrus synchronization are often lower than desired (Odde, 1990; Kesler and Favero, 1996). Factors that negatively affect the establishment of pregnancy include lack of synchronization (Burns et al., 1993; Kesler and Favero, 1996; Kesler et al., 1997a), subnormal luteal phases (Kesler and Favero, 1996), and embryonic/fetal mortality (Ayalon, 1978; Diskin and Sreenan, 1980; Roche, 1981). Studies have demonstrated that supplemental progestins (progesterone and norgestomet) during the luteal phase may enhance the establishment of pregnancy to the previous insemination (Johnson et al., 1958; Robinson et al., 1989; Favero et al., 1993). Further, Kesler (1997) demonstrated that norgestomet implants maintain pregnancy to term in heifers ovariectomized 10 d after insemination. We hypothesized that norgestomet implants administered five to 24 d after AI would allow cows to maintain embryos in the absence of corpora lutea and permit the determination, via transrectal ultrasonography, of embryo loss due to corpus luteum regression. Additional objectives were to determine if norgestomet implants would 1) facilitate the establishment of pregnancy in cows with short luteal phases, and 2) enhance the establishment of pregnancy. MATERIALS AND METHODS Angus and crossbred beef cows from the University of Illinois Beef Unit (Urbana; n = 52) and the Orr Beef Center (Baylis, IL; n = 86) were included in this experiment. Cows were 12 to 106 d postpartum at the time of the first AI and were fed alfalfa and fescue hay or grazed legume and grass pasture and fed a complete vitamin and mineral mixture to meet NRC requirements (NRC, 1996). Cows at both locations received booster vaccines against infectious bovine rhinotracheitis, bovine virus diarrhea, parainfluenza 3, campylobacteriosis, and leptospira canicola-grippotyphosa-hardjoicterohaemorrhagiae-pomona (Preg-Guard 9 at Urbana and Cattlemaster 4 + VL5 at Baylis) 21 d before the first AI. The vaccine used at Baylis also contained bovine respiratory syncytial virus vaccine. Cows were bled 10 d before and immediately before the administration of Syncro-Mate B (SMB; Merial, Athens, GA) which was administered to all cows to synchronize estrus. The SMB procedure consists of an intramuscular injection of norgestomet (3.0 mg) and estradiol valerate (5.0 mg) in sesame oil and a subcutaneous 6.0-mg norgestomet implant on the convex surface of the ear (Kesler and Favero, 1995). After nine d, SMB implants were removed and cows were artificially inseminated about 48 h later with commercially frozen semen. Five d after the 48-h AI, the cows were randomly assigned to two groups. Treated cows (Urbana, n = 26; Baylis, n = 43) were implanted with two norgestomet/silicone implants that were subcutaneously inserted into the convex surface of the ear. Control cows (Urbana, n = 26; Baylis, n = 43) were untreated but were moved through the chute as the treated cows. The norgestomet/silicone implants were 3.45 mm in diameter and 20 mm in length. Previous research demonstrated that two of these implants release more than 2.5 times a dosage of norgestomet that suppresses estrus (Kesler et al., 1995; Machado and Kesler, 1996) and an amount of norgestomet that maintains pregnancy in ovariectomized heifers (Kesler, 1997). The norgestomet/silicone implants were removed 10 d after implantation and two new norgestomet/silicone implants were administered and left in situ until 24 d after the 48-h AI.

3 Additional blood samples were collected from the cows immediately after SMB implant removal, at the 48-h AI, and 3, 6 or 7, 9 or 10, 13, 16 or 17, 20, 24, 27, 30 or 31, 34, and 38 d after the 48-h AI. All blood samples were collected via jugular venipuncture into syringes using 18 g needles that were 3.81 cm long. Blood samples were immediately placed in an ice water bath until centrifugation at 2,000 x g for 10 min at 4 o C (Wiseman et al., 1983). Serum was separated and stored in one ml vials at - 20ºC until assayed. Progesterone concentrations were determined by a validated ELISA (Kesler et al., 1990). All cows were observed for estrus twice daily, morning and evening, for 31 d beginning the day after SMB implant removal. Standing to be mounted by other cows was the criterion used to determine estrus. Cows in estrus from 72 h to 31 d after SMB implant removal were bred via AI approximately 12 h after the detection of estrus. Commercially frozen semen was used for all artificial inseminations and service sire was chosen before cows were randomly assigned to groups. Fertile bulls were then included with the cows on d 31. Cows with progesterone 1.5 ng/ml in either one or both of the two blood samples collected before SMB treatment were considered to be estrus-cycling. Cows with < 1.5 ng/ml in both blood samples collected before SMB treatment were considered anestrus. Cows determined not to be synchronized with SMB (cows with progesterone concentrations 1.0 ng/ml at the 48-h AI and 3 d later) were eliminated from the study. Cows were examined for pregnancy via transrectal ultrasonography using an ultrasound with a 7.5 MHZ linear array transducer 24 and 38 d after the 48-h AI (Pierson et al., 1988). Thirty-five of 36 cows (97%) pregnant on d 38 were identified as pregnant on d 24 similar to results of Pierson et al. (1988). First service pregnancy rate was based on transrectal ultrasonography determination of pregnancy on d 38. Cows were classified as calving from an insemination if they calved 283 ± 10 d later. Thirtyfour of the 36 (94%) cows diagnosed pregnant by transrectal ultrasonography calved 283 ± 10 d later. One of the two diagnosed pregnant by transrectal ultrasonography that did not calve aborted about six mo after insemination as a result of anaplasmosis. The loss of pregnancy in the other cow was not observed. Second service pregnancy rate was based on calving. Because location had no effect (P >.10), it was not included in the results. Qualitative data were analyzed by chi-square analysis (Cochran and Cox, 1957). Pregnancy rate was also analyzed as a 2 x 2 x 2 factorial split-plot analysis of variance design with treatment (control and norgestomet treated), estrous cycles (anestrus and estrus-cycling), and time (d 24 and d 38) as the main effects (Gill and Hafs, 1971). Progesterone concentrations were analyzed as a 2 x 3 x 6 factorial split-plot analysis of variance design with treatment (control and norgestomet treated), pregnancy status (pregnant from the first AI [38 d], pregnant on d 24 but not on d 38, and not pregnant from the first AI), and time (d 0 to 16 or 17) as the main effects (Gill and Hafs, 1971).

4 RESULTS Norgestomet implants increased (P <.01) the 24 d pregnancy rate (pregnancy rates = 30% and 57% for control and norgestomet treated cows, respectively). The 24 d pregnancy rate (54%) for the previously anestrus cows administered norgestomet was not different (P >.20) from the untreated estrus-cycling cows. The 24 d pregnancy rate for the norgestomet treated cows with progesterone concentrations indicative of cows with subnormal luteal phases (60%) was not different (P >.20) from the pregnancy rate of untreated estrus-cycling cows (41%). The pregnancy rate decreased (P <.05) from d 24 to 38 in the norgestomet treated cows; however, the pregnancy rate on d 38 for the previously anestrus cows administered norgestomet was greater (P <.05) than for the untreated previously anestrous cows. Although norgestomet implants maintained pregnancy in the absence of corpora lutea, embryonic loss occurred after norgestomet implant removal on d 24 in the absence of corpora lutea. CONCLUSION These data demonstrate that the pregnancy rate in norgestomet treated cows after their first postpartum ovulation was equivalent to the pregnancy rate of untreated cows beyond their first postpartum ovulation. Furthermore, the pregnancy rate in norgestomet treated cows with progesterone concentrations of cows with subnormal luteal phases was equivalent to cows with normal estrous cycles. LITERATURE CITED Ayalon, N A review of embryonic mortality in cattle. J. Reprod. Fertil. 54, 483. Burns, P. D., Spitzer, J. C., Bridges, Jr., W. C., Henricks, D. M., and Plyler, B. B., Effects of metestrous administration of a norgestomet implant and injection of norgestomet and estradiol valerate on luteinizing hormone and development and function of corpora lutea in suckled beef cows. J. Anim. Sci. 71, 983. Cochran, W. G. and Cox, G. M., Experimental Design (2nd ed.). John Wiley & Sons, New York. Diskin, M. G. and Sreenan, J. M., Fertilization and embryonic mortality rates in beef heifers after artificial insemination. J. Reprod. Fertil. 59, Favero, R. J., Faulkner, D. B., and Kesler, D. J., Norgestomet implants synchronize estrus and enhance fertility in beef heifers subsequent to a timed artificial insemination. J. Anim. Sci. 71, Gill, J. L. and Hafs, H. D., Analysis of repeated measurements of animals. J. Anim. Sci. 33, 331. Johnson, K. R., Ross, R. H., and Fourt, D. L., Effect of progesterone administration on reproductive efficiency. J. Anim. Sci. 17, 386.

5 Kesler, D. J Norgestomet implants maintain pregnancy in ovariectomized heifers. Theriogenology 48, 89. Kesler, D. J., Dyson, T. S., Summers, R. N., Steckler, T. L., and Nash, T. G., 1997a. Effect of prostaglandin F 2α treatment before norgestomet and estradiol valerate treatment on regression, formation, and function of corpora lutea in beef heifers. Anim. Reprod. Sci. 47, 281. Kesler, D. J. and Favero, R. J., Estrus synchronization in beef females with norgestomet and estradiol valerate. Part 1: mechanism of action. Agri-Pract. 16, 6. Kesler, D. J. and Favero, R. J., Estrus synchronization in beef females with norgestomet and estradiol valerate. Part 2: factors limiting and enhancing efficacy. Agri-Pract. 17, 12. Kesler, D. J., Favero, R. J., and Troxel, T. R., A comparison of hydron and silicone implants in the bovine norgestomet and estradiol valerate estrus synchronization procedure. Drug. Dev. Ind. Pharm. 21, 475. Kesler, D. J., Khazali, H., and Favero, R. J., Quantification of steroids via a polymer linked second antibody immunoassay: methods of linking anti-rabbit IgG to poly(styrene). In: Progress in Biomedical Polymers. Ed., C.G. Gebelein and R. Dunn. Plenum Publishing Co., New York, p Machado, R. and Kesler, D. J., The efficacy of norethindrone acetate and norgestomet implants in suppressing estrus in beef cows. Drug Dev. Ind. Pharm. 22, NRC Nutrient Requirements of Beef Cattle. National Academy Press, Washington, DC. Odde, K.B A review of synchronization of estrus in postpartum cattle. J. Anim. Sci. 68, 817. Pierson, R. A., Kastelic, J. P., and Ginther, O. J., Basic principles and techniques for tranrectal ultrasonography in cattle and horses. Theriogenology 29, 3. Robinson, N. A., Leslie, K. E., and Walton, J. S., Effect of treatment with progesterone on pregnancy rate and plasma concentrations of progesterone in Holstein cows. J. Dairy Sci. 72, 202. Roche, J. F Reproductive wastage following artificial insemination of heifers. Vet. Record 109, 401. Wiseman, B. S., Vincent, D. L., Thomford, P. J., Scheffrahn, N. S., Sargent, G. F., and Kesler, D. J., Changes in porcine, ovine, bovine and equine blood progesterone concentrations between collection and centrifugation. Anim. Reprod. Sci. 5, 157.

6 Table 1. Effect of norgestomet on pregnancy rate, P 4 concentrations, and embryonic loss Control Norgestomet Treated P < Pregnancy Rate-d 24 (anestrus) 1/20 ( 5%) a 14/26 (54%) cef.01 Pregnancy Rate-d 38 (anestrus) 0/20 ( 0%) b 7/26 (27%) d.01 Pregnancy Rate-d 24 (cyclic) 18/44 (41%) ae 21/36 (58%) c.13 Pregnancy Rate-d 38 (cyclic) 15/44 (34%) b 14/36 (39%) d NS P 4 ng/ml (d 6 or 7): pregnant 3.77 ±.74 g 3.49 ±.56 g ---- embryonic loss 1.66 ±.79 g 2.62 ±.42 g ---- Pregnant (d 38)/pregnant (d 24): < 1.0 ng/ml P 4 d /0 ( 0%) 0/3 ( 0%) ---- < 1.5 ng/ml P 4 d 20 0/0 ( 0%) 0/8 ( 0%) ---- > 1.0 d 6 & > 1.5 ng/ml P 4 d 20 15/19 (79%) 21/24 (88%) NS a,b Values with similar superscripts differ (P <.01). c,d,e Values with similar superscripts are not different (P >.25). f The pregnancy rate for norgestomet treated cows that ovulated and had short luteal phases was 50% (3/6) which was similar to the pregnancy rate on d 24 for the cyclic cows (41%). g Cows with embryonic loss had lower (P <.05) P 4 concentrations than pregnant cows.