Uterine contraction patterns and fertility in early postpartum ewes

Small Ruminant Research 38 (2000) 51±56 Uterine contraction patterns and fertility in early postpartum ewes D.O. Kiesling *, M.A. Akinbami 1, S. Meredith, J.E. Warren Jr 2 307 Foster Hall, Lincoln University, Jefferson City, MO 65102-0029, USA Received 26 August 1999; accepted 23 February 2000 Abstract Three experiments were conducted to determine: (1) the direction of uterine contractions in Days 32 and 52 postpartum ewes (Experiment 1); (2) the effect of PGF 2a on direction of uterine contractions (Experiment 2); and (3) the effect of PGF 2a on fertility rates in Day 32 postpartum ewes (Experiment 3). In Experiment 1, non-lambing (>90 days postpartum) and lambing ewes (day of lambingˆday 0) received medroxyprogesterone acetate (MAP) vaginal sponges for 8 days and 500 IU of ecg at sponge withdrawal (Days 30 or 50 postpartum). At the time of ecg injection, ewes were divided into the following groups: (1) non-lambing (control; nˆ29); (2) Day 32 postpartum dry (nˆ15) and lactating (nˆ16); and (3) Day 52 postpartum dry (nˆ14) and lactating (nˆ16). At estrus or 60 h post-ecg, the uterus was exteriorized through a mid-ventral incision, and the origin and direction of uterine contractions were recorded for 10 min. In Experiment 2, ewes received MAP sponges on Day 16 postpartum followed by 500 IU of ecg on day of sponge removal (Day 30). At estrus, the ewes were divided into the following treatments: (1) two injections of saline 4 h apart (nˆ10) and (2) 12.5 mg of PGF 2a followed by another 12.5 mg 4 h later (nˆ7). After the second injection, ewes were laparotomized and uterine contractions were counted. In Experiment 3, estrus was induced in postpartum ewes, and ewes were mated to two rams, then received the same two treatments as described in Experiment 2 (ram saline; nˆ32 and ram PGF 2a ; nˆ28). Two days following mating, ewes were laparotomized and the oviducts ushed for recovery of ova. In Experiment 1, lactational status had no effect, therefore, the data were pooled. Control ewes had a greater percentage (p<0.05) of uterine contractions (69%) moving towards the oviducts than did Day 32 (8%) or Day 52 (43%) ewes. In Experiment 2, PGF 2a treatment increased the proportion of contractions (p<0.05) moving toward the oviducts (controls 16%, PGF 2a 42%). Number of PGF 2a -treated ewes (Experiment 3) with fertilized ova were not signi cantly different from the control ewes (5/32 versus 2/28; respectively). In conclusion, it can be said that the direction of uterine contractions moving toward the oviducts increased as the postpartum interval progressed or if they received PGF 2a injection. PGF 2a treatment did not improve fertility rates in Day 32 postpartum ewes. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Fertility rates; PGF 2a ; Postpartum ewes; Uterine contractions 1. Introduction * Corresponding author. Tel.: 1-573-681-5384; fax: 1-573-681-5548. E-mail address: kiesling@lincolnu.edu (D.O. Kiesling) 1 Present address: Morehouse School of Medicine, Atlanta, GA 30310-1495, USA. 2 Present address: West Virginia University, Morgantown, WV, USA. In an estrous ewe the majority of uterine contractions originate from the caudal part of the uterine horn and move cranially (Hawk, 1973; Hawk and Echternkamp, 1973). This contraction pattern has been associated with the rapid transport of sperm to the oviducts (Hawk, 1983). It is not known if the direction of uterine contractions are the same in ewes at an induced 0921-4488/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0921-4488(00)00138-3

52 D.O. Kiesling et al. / Small Ruminant Research 38 (2000) 51±56 estrus during the early postpartum period. Ewes bred at an induced estrus 32 days after lambing have poor fertility (10%). Fertility improves as lambing to breeding interval increases with an 80% conception rate on Day 52 postpartum (Warren et al., 1989). The low fertility observed in ewes during the early postpartum period has been associated with poor sperm transport (Warren et al., 1989). In rabbits, prostaglandin F 2a, (PGF 2a ) injected near the time of insemination increased the sperm numbers in the oviducts of the reproductive tract 2±3 h later (Hawk et al., 1982). In cycling ewes, injections of PGF 2a increased both frequency and strength of uterine contractions as measured by a physiograph (Hawk and Conley, 1985). Edquist et al. (1975) and Gustafsson (1978) injected PGF 2a into ewes at the time of insemination and found more sperm in the oviducts of treated than of control ewes 16 or 24 h later. Dimov and Georgiev (1977) demonstrated that the addition of PGF 2a, and prostaglandin E 2 to diluted ram semen increased fertility by more than 15%. To achieve two lamb crops in 1 year, acceptable fertility rates must be obtained within 30±35 days after lambing. It appears that sperm transport to the site of fertilization is minimal in 32 days postpartum ewes. Therefore, factors that could in uence sperm transport such as direction of uterine contractions and the effect of PGF 2a on uterine contractions and fertility need to be evaluated. The following experiments were designed to: (1) determine the pattern of uterine contractions of Days 32 and 52 postpartum ewes, compared to non-lambing controls; (2) evaluate the effect of PGF 2a on direction of uterine contractions in the postpartum ewe and (3) determine the effect of PGF 2a on fertility in Day 32 postpartum ewes bred during the anestrous and estrous seasons. 2. Materials and methods 2.1. Animals HampshireSuffolk cross ewes (nˆ202) ranging in age from 2 to 5 years were maintained in a dry lot with access to a barn with an open southern exposure. Ewes were fed a 12% CP corn±soybean meal ration (0.25 kg per ewe per day) plus orchard/grass hay ad libitum. The experiments were conducted in the Midwestern region of the United States near Jefferson City, MO, located at 388N latitude, 948W longitude and at a geographical altitude of 168 m. 2.2. Experiment 1 Ewes from June and July lambing, and non-lambing ewes (controls; at least 90±120 days from last lambing; nˆ29) were used for the study. Beginning on Days 22 or 42 postpartum (day of lambingˆ0) ewes received a 40 mg medroxyprogesterone acetate (MAP; Intervet, Holland) impregnated vaginal sponge for 8 days. At sponge removal ewes were injected with 500 IU ecg (Intervet, Holland). Estrus was induced in controls using the same procedure except the time that treatment was initiated was no earlier than 90±120 days postpartum. Ewes were randomly assigned within postpartum days into the following groups: (1) Day 32 postpartum dry (nˆ15) and postpartum lactating (nˆ16) and (2) Day 52 postpartum dry (nˆ14) and postpartum lactating (nˆ16). Postpartum dry ewes either lost their lambs at birth or had their lambs weaned at Day 10 postpartum. Lactating ewes had their lambs with them throughout the experiment. At estrus or 60 h post-ecg (whichever came rst), ewes were anesthetized with 20 mg of pentobarbital sodium, placed in dorsal recumbency on a surgical table, and the uterus exteriorized through a mid-ventral incision. Uterine contractions were observed using the procedure of Hawk (1975). Three minutes after the uterus was exteriorized, the origin and direction of movement of uterine contractions was recorded during a 10 min interval (by an observer for each horn). Contractions were recorded as originating either in the caudal, middle or cranial one-third of the uterine horn. Direction of contractions was recorded as either moving toward the oviduct, toward the cervix, in both directions, or no movement. The uterus was not moistened during the period of observation to prevent stimulus-induced contractions. Drying of the serosa was not observed. The initial analysis was a 22 factorial arrangement with two treatments (Days 32 and 52 postpartum) and two different lactation states (lactating and non-lactating). Dependent variables tested in the model were direction and origin of uterine contractions. There was no effect of lactational status, therefore the data were reanalyzed as a one-way analysis of

D.O. Kiesling et al. / Small Ruminant Research 38 (2000) 51±56 53 variance (ANOVA) with three treatments: controls, Day 32 postpartum and Day 52 postpartum ewes. Least square means were performed to determine differences between speci c treatments (SAS, 1988). Percentage of ewes exhibiting estrus, was determined by chi-square (SAS, 1988). 2.3. Experiment 2 Ewes (nˆ17) which lambed in September and October each received a vaginal MAP sponge on Day 16 postpartum (day of lambingˆday 0) followed by 500 IU of ecg on day of sponge removal (Day 30). At estrus, the ewes were divided into the following treatment groups: (1) controls received two injections of 0.9% saline (2.5 ml each) 4 h apart (nˆ10) and (2) PGF 2a -treated ewes (nˆ7) received two injections (each injection 12.5 mg) of PGF 2a 4 h apart (Lutalyse, Pharmacia & UpJohn Co., Kalamazoo, MI, USA). Immediately following the second injection, ewes were laparotomized and uterine contractions were recorded in each horn for a 10 min period as described in Experiment 1. Differences between treatments in direction of uterine contractions were determined in ANOVA (SAS, 1988). 2.4. Experiment 3 Postpartum ewes (nˆ20) which lambed in September and October, and postpartum ewes (nˆ40) which lambed in February and March, received a vaginal MAP sponge on Day 16 postpartum (day of lambingˆday 0) and 750 IU ecg at the time of sponge removal on Day 30 postpartum. Ewes that were 90± 120 days since last lambing (nˆ15, estrous season; nˆ20, anestrous season) were mated at an induced estrus (received a MAP sponge for 14 days plus 750 IU ecg at sponge removal) and allowed to lamb to determine fertility of rams. Ewes were checked for estrus twice daily with a vasectomized ram. At estrus, postpartum ewes were randomly assigned to two treatment groups: (1) ram saline, which were mated by two fertile rams during a 10 min period and given an injection of 2.5 ml of 0.9% saline after mating and an additional injection 4 h later (nˆ28) and (2) ram PGF 2a -treated ewes, which were mated as described in Group 1 and given an injection of 12.5 mg of PGF 2a immediately after mating and an additional 12.5 mg injection 4 h later (nˆ32). Two days following estrus, postpartum ewes were laparotomized and the oviduct adjacent to the ovary containing the corpus luteum was ushed for ova. Ova were evaluated for cleavage under a phase contrast microscope. The initial design was a 22 factorial arrangement with two treatments (ram saline and ram PGF 2a ) and two seasons (fall and spring). There was no effect of season, therefore, the data were pooled and reanalyzed as ANOVA with two treatments (ram saline and ram PGF 2a ) using the General Linear Models Procedure of SAS (SAS, 1988). 3. Results 3.1. Experiment 1 Percentage of ewes exhibiting estrus was similar among controls, Days 32 and 52 ewes (69, 55 and 50%), respectively. Mean number of uterine contractions moving toward the oviducts was greater (p<0.05) for the control ewes than the Days 32 or 52 ewes (Table 1). Day 52 ewes had more contractions (p<0.05) moving towards the oviducts than Day 32 ewes. There were more uterine contractions moving towards the cervix of Day 32 ewes than in Day 52 ewes. For the control ewes, origin of contractions was equally divided among the caudal, middle and cranial portions of the uterine horn (Table 1). The majority (76%) of the uterine contractions originated from the cranial part of the uterus and moved towards the cervix in Day 32 ewes, whereas only 51% of uterine contractions originated in the cranial part of the uterus and moved toward the cervix in Day 52 ewes (Table 1). 3.2. Experiment 2 Since there were no differences among treatments in number of uterine contractions moving both ways in Experiment 1, only contractions moving towards or away from oviducts were recorded in this experiment. There was no signi cant difference in the number or direction of contractions between uterine horns, so the data recorded were pooled. Percentage of ewes exhibiting estrus was 100% in both treatment groups.

54 D.O. Kiesling et al. / Small Ruminant Research 38 (2000) 51±56 Table 1 Direction and origin of uterine contractions (means.e.) in postpartum ewes a Uterine contractions Treatment b Control (nˆ29) Day 32 (nˆ31) Day 52 (nˆ30) Total number of contractions 322 362 353 Direction c Moving towards oviducts 222 (69%) c 31 (8%) d 152 (43%) e Moving neither or both ways 21 (6%) 31 (8%) 31 (8%) Moving towards cervix 81 (25%) c 302 (84%) d 172 (49%) e Origin d Caudal 122 (38%) c,e 31 (8%) d 101(28%) e Middle 111 (33%) 61 (16%) 71 (21%) Cranial 91 (28%) c 272 (76%) d 182 (51%) e a Numbers in rows with different letters (c, d, e) differ (p<0.05). b Uterine contractions of control ewes originated throughout the uterus and were moving towards the oviducts, whereas, Days 32 and 52 ewes majority of uterine contractions originated in the anterior part of the uterus and moved toward the cervix. c Mean uterine contractionss.e. per 10 min. Percentages are of the total contractions moving towards or away from oviducts. d Percentages are of the total contractions originating from the caudal, middle or cranial part of the uterus. Table 2 Effect of PGF 2a on uterine contractions in Day 32 postpartum ewes Treatment N Mean uterine contractionss.e. per 10 min Total Moving towards oviducts a Away from oviducts a Control 10 321 51 (16%) a 271 (84%) a PGF 2a 7 242 102 (42%) b 142 (58%) b a Numbers in columns with different letters (a, b) differ (p<0.05). PGF 2a -treated ewes had more (p<0.05) contractions moving towards the oviducts than did the controls (Table 2). The percentage of contractions moving toward the cervix was 58% (p<0.05) for PGF 2a -treated ewes compared to 84% in the controls. 3.3. Experiment 3 Table 3 Effect of PGF 2a on fertility in Day 32 postpartum ewes a Treatment N Ewes with fertilized ova Ewes with unfertilized ova Ram saline 28 2 (7%) 26 (93%) Ram PGF 2a 32 5 (16%) 27 (84%) a No difference between treatment or season (p>0.05). The percentage of ewes exhibiting estrus was 100% in all treatment groups. The percentage of ewes lambing was 66% for controls (90±120 days since last lambing) indicating normal fertility of rams used in this study. PGF 2a treatment of Day 32 postpartum ewes did not improve fertility (Table 3). 4. Discussion Some reports have suggested that the uterus has completed involution in ewes by 30 days after lambing (a review by Kiracofe, 1980). However, histological evaluation of the luminal contents and endometrium at this time revealed that the number of lymphocytes and plasma cells exceeded those found in control ewes (Akinbami, 1989). The amount of uterine debris and leukocytes decreases as the time from lambing increases, with a corresponding increase in contractions moving from the cervix to the oviducts, and an increase in fertility rate.

D.O. Kiesling et al. / Small Ruminant Research 38 (2000) 51±56 55 This is supported by data demonstrating that placement of intrauterine devices (IUD) into the uterine horn of estrous ewes causes contraction patterns similar to Day 32 postpartum ewes. This reversal of direction of contractions with IUD resulted in a reduced number of sperm in the oviducts and is associated with loss of fertility (Hawk, 1967, 1969). Once the device is removed, direction of contractions move from the cervix to the oviduct and fertility is restored. PGF 2a was injected in an attempt to change direction of contractions in Day 32 postpartum ewes. Although PGF 2a increased the number of uterine contractions moving towards the oviducts, there was no improvement in fertility. Even with the change in direction of contractions, sperm may not have been transported in large enough numbers to the oviducts for fertilization to take place (Hunter and Nichol, 1983). The effect of changing the direction of uterine contractions on the number of sperm actually reaching the site of fertilization was not determined in the present experiment. It seems unlikely, however, that PGF 2a successfully increased the number of sperm at the ampullary-isthmus junction because surgical deposition of semen at this site signi cantly improved fertility in Day 32 postpartum ewes (Warren et al., 1989). Even if the change in direction of uterine contractions by PGF 2a resulted in more sperm moving towards the site of fertilization, they were probably destroyed in the uterus before reaching the site. This is thought possible because of the higher number of leukocytes found in the Day 32 postpartum uterus (Akinbami, 1989) and leukocytes are one of the two major causes for loss of sperm (Bedford, 1965; Mattner, 1968). The adverse uterine environment at this postpartum stage is further illustrated by Akinbami et al. (1996) and Wallace et al. (1989) who demonstrated that embryos migrating into the uterine horn have poor survival rate early postpartum. Future treatments to improve fertility may need to be focused more on improving the uterine environment rather than direction of uterine contractions. 5. Conclusion The high proportion of uterine contractions originating near the oviducts and moving toward the cervix in Day 32 postpartum ewes could be one cause for the poor fertility in ewes bred during the postpartum period. Although direction of uterine contractions could be reversed with PGF 2a injections, there was no improvement in fertility. Evaluating factors that cause low fertility during the early postpartum period may result in methods to improve fertility at this stage. Acknowledgements The authors are grateful to G.A. Dudenhoeffer and Arlene Stewart for their assistance with the handling of ewes. This work has been supported by CSREES project No. MO-X-OP96±416 of the Animal Science Program in Cooperative Research. References Akinbami, M., 1989. Maternal factors affecting conception in postpartum ewes. Ph.D. Dissertation, University of Missouri, Columbia. Akinbami, M.A., Kiesling, D.O., Meredith, S., Warren Jr., J.E., Day, B.N., 1996. Embryonic survival in the uterus of ewes inseminated at the uterotubal junction on day 32 postpartum. Theriogenology 45, 1129±1139. Bedford, J.M., 1965. Effect of environment on phagocytosis of rabbit spermatozoa. J. Reprod. Fertil. 9, 249±256. Dimov, V., Georgiev, G., 1977. Ram semen prostaglandin concentration and its effect on fertility. J. Anim. Sci. 44, 1050±1054. Edquist, S., Einarsson, S., Gustafsson, B., 1975. Effect of prostaglandin F 2a on sperm transport in the reproductive tract of the ewe. Acta Vet. Scand. 16, 149±151. Gustafsson, B.J., 1978. Aspects of fertility with frozen-thawed ram semen. Cryobiology 15, 358. Hawk, H.W., 1967. Investigations on the anti-fertility effect of intrauterine devices in the ewe. J. Reprod. Fertil. 14, 49±59. Hawk, H.W., 1969. Some effects of intrauterine devices on reproductive function in the ewe. Fertil. Steril. 20, 1±13. Hawk, H.W., 1973. Uterine motility and sperm transport in the estrous ewe after prostaglandin-induced regression of Corpora lutea. J. Anim. Sci. 37, 1380±1385. Hawk, H.W., 1975. Hormonal control of changes in the direction of uterine contractions in the estrous ewe. Biol. Reprod. 12, 423± 430. Hawk, H.W., 1983. Sperm survival and transport in the female reproductive tract (cattle, sheep, swine). J. Dairy Sci. 66, 2645± 2660. Hawk, H.W., Conley, H.H., 1985. Effect of prostaglandin F 2a, phenylephrine and ergonovine on uterine contractions in the ewe. J. Anim. Sci. 60, 537±543.

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