Comparison of Gravid Uterine Parameters in Naturally Bred Ewes and Ewes after Transfer of In Vitro Produced Embryos, and in Single, Twin and Triplet Pregnancies

Large offspring syndrome (LOS) may be initiated in ovine embryos after exposure to in vitro conditions. The objective of this study was to compare various parameters of the gravid uterus on day 140 after fertilization: (i) in ewes (n=4) bred naturally, and in ewes (n=18) after transfer of embryos produced in vitro, and (ii) in single, twin and triplet pregnancies after transfer of in vitro produced embryos. On day 5 after in vitro fertilization, two to three embryos with 16 or more cells were transferred to recipient ewes that were at day 5 after estrus. Pregnancy was verified by real-time ultrasonography on day 45 or later after breeding or embryo transfer (ET). On day 140 of pregnancy, the reproductive tract was collected from all ewes and the following parameters were determined: the number, sex and weight of fetuses, weight of uterus and fetal membranes, weight and numbers of placentomes. Weight of fetuses was similar for naturally bred ewes and ewes after ET, but the weight of individual placentomes was greater (P<0.05) for ewes after ET (8.5±0.5 g) than for naturally bred ewes (5.4±1.9 g) and the total weight of placentomes/ewe tended to be greater (P<0.1) for ewes after ET (0.66±0.05 kg) than for the naturally bred ewes (0.48±0.03 kg). The number of placentomes/ewe was less (P<0.01) for single pregnancies (67.0±4.2) than for twin (87.0±4.1) or triplet (85.7±3.7) pregnancies. The number of placentomes/fetus was greater (P<0.01) for single (67.0±4.2), less for twin (43.5±2.0) and least for triplet (28.5±1.2) pregnancies. However, the weight of each individual placentome was greater (P<0.06) for triplet (11.2±1.2 g) than for twin (8.3±0.8 g) or single (7.7±0.5 g) pregnancies. For single, twin and triplet pregnancies, the number of fetuses per ewe was negatively correlated with the weight of placentomes/fetus (r= -0.65; P<0.003), the number of placentomes/fetus (r = -0.86; P<0.001) and the weight of the fetus (r = -0.80; P<0.001) and positively correlated with the weight of individual placentome (r= +0.50; P<0.03). These data demonstrate the lack of large offspring syndrome, indicating that culture conditions were optimal for production of ovine embryos. The low number of placentomes/fetus seen in multiple pregnancies appears to be compensated for by the increase in the total number of placentomes and the weight of each placentome. Introduction In vitro production of embryos from domestic animals is used to augment conventional genetic improvement programs in agriculture and for research purposes. However, the adoption of advanced reproductive technologies for embryo production and transfer in ruminants may result in the occurrence of large offspring syndrome (LOS) also termed as fetal oversize syndrome (Young et al., 1998; Walker et al., 1996; Thompson et al., 1997; Farin et al., 2001). This syndrome has been associated with increased dystocia and abnormal growth and development at fetal, neonatal and later stages in life. A number of treatments have been shown to cause this perturbation including in vitro culture of embryos and transfer of embryos to recipients (Young et al., 1998; Walker et al., 1996; Thompson et al., 1997; Farin et al., 2001). This emphasizes the need to optimize culture conditions to simulate natural uterine environment. Several studies have demonstrated the effects of single, twin and triple pregnancies on fetal weight (Brown et al., 1998; Reynolds and Redmer, 1995), but parameters of the gravid uterus have not been evaluated in detail. The objective of this study was to compare various parameters of the gravid uterus on day 140 after fertilization: (i) in ewes bred naturally, and in ewes after transfer of embryos produced in vitro, and (ii) in single, twin and triplet pregnancies after transfer of in vitro produced embryos. Material and Methods Non-pregnant crossbred range ewes were divided into two groups, one group was allowed to breed naturally (n=4) and the other (n=18) was subjected to embryo transfer (ET). 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 to collect ovaries (Reynolds et al., 1998). Oocytes were collected, matured and fertilized in vitro by frozenthawed semen (Stenbak et al, 2001; Grazul-Bilska et al., 2003). Embryos were cultured for 5 days and then transferred at a stage of 16 or more cells to recipient ewes 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 i.m. injection of PGF2α (Estrumate, Schering-Plough Animal Health Corp., Union, NJ., 125 mg/ml/injection) in the morning and evening on day 8-12 of the estrous cycle. Two to three embryos with 16 or more cells were transferred to recipient ewes surgically by placing the embryos in the tip of the uterine horn ipsilateral to the ovary containing a corpus luteum. Four ewes were naturally bred with fertile Hampshire rams. The recipient ewes were placed with vasectomized rams beginning on day 6 after embryo transfer to check estrus. In addition, the presence of fetuses was determined by ultrasonography (Classic Ultrasound Equipment Ltd, Tequesta, FL) on day 45 or later after embryo transfer. On day 140 of pregnancy, recipient ewes and the ewes bred naturally were slaughtered and the gravid reproductive tract was collected. Then the number and weight of fetuses, weight of uterus and fetal membranes, weight and numbers of placentomes were determined. Data were analyzed by using the general linear models (GLM) procedure of the Statistical Analysis System (SAS, 1985). When the F-test was significant, differences between specific means were evaluated using the Duncan test (Kirk, 1982). All data are reported as means ± standard error (SEM). Results Several parameters of the gravid uteri evaluated for the naturally bred ewes and for ET recipient ewes are shown in Table 1. Table 1: Parameters of gravid uteri on day 140 after fertilization for ewes bred naturally and for ewes after transfer of embryos produced in vitro. Pregnancy after Pregnancy after transfer of in vitro Parameter natural breeding produced embryos Number of pregnant ewes 4 18 Weight of gravid 10.48±0.26 13.76±1.13 uterus/ewe (kg) Weight of fluid /ewe (kg) 1.96±0.26 3.13±0.48 Weight of uterus plus fetal 3.11±1.0 2.13±0.13 membranes/ewe (kg) Weight of fetal Not available 0.45±0.05 membranes/ewe (kg) Total weight of 0.48±0.03 0.66±0.05 placentomes/ewe (kg) Total weight of 0.43±0.08 0.42±0.03 placentomes/fetus (kg) Total number of 67.5.0±4.3 77.8±3.4 placentomes/ewe Total number of 57.8±6.8 51.5±4.1 placentomes/fetus Mean weight of 5.43±1.96 a 8.51±0.53 placentome/ewe (g) Mean weight of 5.19±0.78 4.92±0.17 fetus (kg) Total fetal weight/ewe 6.78± 0.25 8.49±0.63 (kg) (5 fetuses) (31 fetuses) Range of fetal weight 3.1-7.1 2.95-6.4 (kg) a,b P<0.05, A,B P<0.10; Values differ within a row. The weight of the gravid uterus, weight of fetal membranes, weight of placentomes/ewe, total number of placentomes, mean weight of fetus and total fetal weight/ewe were similar for the naturally bred ewes and for ewes after ET. However, the mean weight of placentome/ewe was greater (P<0.05) for ewes after ET (5.43±1.96 g) than for the naturally bred ewes (8.51±0.53 g). The total weight of placentomes/ewe tended to be greater (P<0.1) for ewes after ET (0.66±0.05 kg) than for the naturally bred ewes