Gonadotrope function in ovariectomized ewes actively immunized against gonadotropin-releasing hormone (GnRH)

The gonadotrope cells of the ovine anterior pituitary were insulated from hypothalamic inputs by imposing an immunologic barrier generated by active immunization of ovariectomized ewes against gonadotropin-releasing hormone (GnRH) conjugated to keyhole limpet hemocyanin (KLH) through a p-aminophenylacetic acid bridge. All GnRH-KLH animals immunized developed titers of anti-GnRH that exceeded 1:5000. The antisera were specific for GnRH and cross-reacted with GnRH agonists modified in position 10 to an extent that was less than 0.01%. Ewes actively immunized against GnRH-KLH displayed levels of basal and GnRH agonist-induced gonadotropin secretion that were markedly lower (p less than 0.05) than comparable parameters in ewes actively immunized against KLH. In contrast, basal and thyrotropin-releasing hormone (TRH)-induced prolactin (PRL) secretion were not compromised by active immunization. Immunization against the GnRH-KLH conjugate, but not KLH alone, prevented expression of the positive feedback response to exogenous estradiol (E2). Pituitary stores of immunoactive luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were significantly (p less than 0.001) reduced in ewes immunized against GnRH-KLH but stores of PRL were not affected by such immunization. Further, the biopotency of the residual LH stores in tissue of animals from the anti-GnRH group was significantly (p less than 0.05) lower than LH biopotency in anti-KLH animals. Serum levels of LH in anti-GnRH ewes were restored by circhoral administration of a GnRH agonist that did not cross-react with the antisera generated. Pulsatile delivery of GnRH agonist in anti-GnRH ewes significantly (p less than 0.05) elevated serum LH within 48 h and reestablished LH levels comparable to anti-KLH ewes within 6 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of a new pregnancy-associated glycoprotein radioimmunoassay method for the detection of early pregnancy in ewes

The aim of the current study was to describe the use of a pool of different antisera raised against pregnancy-associated glycoproteins (PAGs; purified from both ovine and caprine placentas) for early pregnancy diagnosis in ovine species. Sixty-three pluriparous Sarda ewes (Ovis aries) were synchronized. Blood samples were withdrawn on Days 18, 24, 26, 28, 30, and 50 after mating. These samples were assayed for progesterone (radioimmunoassay [RIA] including an extraction step) and for pregnancy-associated glycoproteins (RIA-706 and RIA-srPool). Progesterone concentrations were under 1.0 ng/mL in all nonpregnant Sarda ewes. In pregnant ewes, mean progesterone concentrations ranged from 2.4 ng/mL (Day 24, single pregnancies) to 4.4 ng/mL (Day 28, multiple pregnancies). During all periods of examination, PAGs remained lower than 0.8 ng/mL in nonpregnant ewes. On Day 18 of pregnancy, PAG concentrations could be detected in 26 of 43 (60.5%) and in 41 of 43 (95.3%) pregnant ewes using the RIA-706 and RIA-srPool methods, respectively. From Day 24 to Day 50, using both RIA methods, PAGs could be detected in all pregnant ewes. On Day 24, the best threshold for pregnancy diagnosis was obtained by use of RIA-srPool, maximal concentration in nonpregnant ewes being 0.3 ng/mL and minimal concentration in pregnant ewes being 4.8 ng/mL. In general, progesterone and PAG concentrations were higher in multiple pregnancies than in single pregnancies. However, because of large individual variations, single pregnancies could not be differentiated from multiple pregnancies.     

Effects of immunization against bone morphogenetic protein 15 and growth differentiation factor 9 on ovulation rate, fertilization, and pregnancy in ewes

Immunization of ewes against growth differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) can lead to an increased ovulation rate; however, it is not known whether normal pregnancies occur following such treatments. The aims of the present study were to determine the effects of a short-term immunization regimen against BMP15 and GDF9 on ovulation rate, fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, and the ability of immunized ewes to carry a pregnancy to term. Ewes were given a primary and booster immunization against keyhole limpet hemocyanin (KLH; control, n = 50), a GDF9-specific peptide conjugated to KLH (GDF9, n = 30), or a BMP15-specific peptide conjugated to KLH (BMP15, n = 30). The estrous cycles of all ewes were synchronized, and ewes were joined with fertile rams approximately 14 days after the booster immunization. The number of corpora lutea was determined by laparoscopy 3-4 days following mating. Subsequently, about one-half of the ewes in each group underwent an embryo transfer procedure 4-6 days following mating, with the embryos being transferred to synchronized, nonimmunized recipients. The remaining ewes were allowed to carry their pregnancies to term. Short-term immunization against either BMP15 or GDF9 peptides resulted in an increase in ovulation rate with no apparent detrimental affects on fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, or the ability of the immunized ewes to carry a pregnancy to term. Therefore, regulation of BMP15, GDF9, or both is potentially a new technique to enhance fecundity in some mammals.     

Plasma urea nitrogen in relation to pregnancy rate in dairy sheep

The aim of this field study was to investigate the relationship of plasma urea nitrogen (PUN) with the pregnancy rate in lactating Awassi × Merino ewes. One hundred and eighty-five Awassi × Merino ewes were used in the present study. Ewes were fed a diet containing 17.4% crude protein and were milked twice a day by the milking machine. The ewes were synchronized for estrus by insertion of intravaginal sponges containing 30 mg flurogestone acetate for 14 days. At the time of sponge removal each ewe was administered eCG (600 IU). All ewes were inseminated twice with fresh semen into the external os of the cervix at 48 and 56 h after sponge removal. The day of insemination was considered as Day 0 for calculating the gestational period. Blood samples were collected from each ewe at Days 0, 18 for measurement of PUN concentrations and at Day 22 after AI for measurement of pregnancy-associated glycoprotein (PAG) by radioimmunoassay (RIA). Thirty-eight ewes (20.5%) were confirmed pregnant by PAG-RIA test at Day 22 and by ultrasonography at Day 80. The mean (±S.D.) concentration of PUN in all ewes at Day 0 was 12.7±4.6 mmol/L. There were non-significant differences in the level of PUN between pregnant and non-pregnant ewes at Days 0 (12.2±4.2 mmol/L vs. 12.8±4.7 mmol/L, respectively) and 18 (9.6±2.9 mmol/L vs. 10.4±4.0 mmol/L, respectively) after AI. Mean PUN concentrations decreased significantly from Day 0 to Day 18 after AI in both pregnant and non-pregnant ewes. By using logistic regression analysis, there was no effect of PUN concentrations on the probability of pregnancy occurrence in the studied ewes (odds ratio: 0.97; 95% confidence interval: 0.9-1.05; P=0.45). In conclusion, there was no evidence of a relationship between PUN concentration and pregnancy rate for lactating Awassi × Merino ewes in the present study because of low pregnancy rate observed.     

A cloning and expression analysis of pregnancy-associated glycoproteins expressed in trophoblasts of the white-tail deer placenta

The pregnancy-associated glycoproteins (PAGs) are placental proteins that have been cloned from swine, sheep, goats, and cattle, but never from animals within the Cervidae family. The goal of this work was to characterize PAGs in white-tailed deer. Placenta and uterine tissues were collected from pregnant does at days 85 and 90 of pregnancy. RNA from cotyledons was used to amplify deer PAGs by RT-PCR. Ten distinct cDNAs were cloned and sequenced. Some normally conserved amino acids comprising the catalytic site were found to be altered in deer PAGs 4, 5, and 8; another PAG, (PAG-9) was a splice variant that lacked exon 7. In each case, these mutations would likely preclude proteolytic activity for these proteins. A phylogenetic analysis revealed that most of the deer PAGs fell within the ancient PAG grouping. The remainder fell within the more modern (BNC-specific) PAG group. Western blotting was performed with anti-PAG antibodies and this analysis revealed that deer PAGs comprise a heterogeneous group based on different antigenicities and electrophoretic mobilities. Immunohistochemistry and in situ hybridization revealed some unique localization patterns of PAGs in the deer placentome compared to those in other ruminants. Most notably, deer PAGs 4 and 5, which according to the phylogeny, are "ancient PAGs," were expected to be present in all trophoblasts; instead, they were localized to the BNC. Although many of the PAGs identified here are very similar to those in Bovidae, some are clearly distinct in their expression pattern and probably possess functional roles unique to cervid reproduction.     

Fertilization and early embryonic development in androstenedione-immunized Merino ewes

Ewes were immunized against androstenedione (Fecundin) and assigned to be mated 14 days (179 ewes Group C) or 25 days (174 ewes Group B) after a booster immunization with Fecundin. The anti-androstenedione titres at these times were 6790 and 3240 respectively (P less than 0.01). The remaining 169 ewes were untreated controls (Group A). Ewes were mated to entire rams (12 rams to 180 ewes) at their second oestrus after synchronization of oestrus. Immunization against androstenedione caused a shortening of the time from sponge removal to mating (Day 0) and a decrease in the percentage of ewes mated by the rams. Also, ovulation rate was increased after immunization (P less than 0.01), being 1.42, 2.16 and 1.93 for Groups A, C and B respectively. Egg recovery rates on Day 2 were lower in immunized ewes and there was some indication that fertilization rates were lowered. On Day 13 after mating a higher proportion of blastocysts was recovered from ewes in Group A than from those in Groups B and C. Immunization resulted in lower fertilization rates and smaller blastocysts with lower mitotic indexes (P less than 0.01). At Days 24-32 of pregnancy fetal weight was lower in the immunized ewes. At all sampling stages, the proportion of ewes pregnant (fertility) was lowered in immunized ewes. The results of the present study show that significant reproductive wastage occurs in androstenedione-immunized Merino ewes, with lower rates of embryo recovery and delayed embryonic development being found in comparison to controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers

The pregnancy-associated glycoproteins (PAGs) are a large gene family expressed in trophoblast cells of ruminant ungulates. The detection of PAGs (more specifically, PAG-1) in maternal serum has served as the basis for pregnancy detection in cattle. Unfortunately, PAG-1 and/or antigenically-related PAGs exhibit a long half-life in maternal serum (>8 d) and can be detected 80-100 d post-partum, thereby producing false positives in animals bred within 60-d of calving. The goal of the present studies was to develop a monoclonal-based assay that targeted early-pregnancy PAGs whose persistence in maternal serum post-partum might be relatively short-lived. Three anti-PAG monoclonal antibodies that recognized distinct subsets of PAGs were selected and used as trapping reagents in a 'sandwich' type of enzyme-linked immunosorbant assay (ELISA). A polyclonal antiserum with broad specificity was used for detecting bound PAGs. A total of 42 cows and heifers were bled daily on day 15, days 22 to 28, and then weekly throughout pregnancy and for 10 weeks (approximately 70 d) into the post-partum period. The ELISA was able to detect PAG in maternal serum of all animals unambiguously by day 28 post-insemination (PAG concentration: 8.75 +/- 3.04 ng/mL). In maternal serum, PAG concentrations peaked during the week of parturition at 588.9 +/- 249.9 ng/mL, and after calving, PAG was completely cleared (half-life: 4.3 d) by eight-week post-partum in 38 of 40 of the animals tested and was at very low concentrations in the remaining two (1.4 and 4.9 ng/mL, respectively). In summary, a monoclonal-based assay has been established that is sensitive enough to detect PAG in maternal serum by the forth week of pregnancy, but does not suffer from carry-over of antigen from a previous pregnancy.     

Active immunization of ewes against ovine placental lactogen increases birth weight of lambs and milk production with no adverse effect on conception rate

In two experiments, 16 Booroola-Assaf and 35 Assaf ewe-lambs were actively immunized at 5 months of age against recombinant ovine placental lactogen (oPL). At 9 months of age, the ewe-lambs were mated for the first time and then introduced into a frequent mating-system. Anti-oPL antibody titers, reproductive performance, maternal serum levels of oPL during pregnancy, lamb birth weight and milk production of the ewes were followed in the immunized ewes and in their non-immunized control counterparts. All the immunized ewes developed anti-oPL antibodies, which interfered with oPL bioactivity in an in vitro cell proliferation assay. Conception rates did not differ (P>0.05) between immunized and non-immunized ewes. Abundant antibody-bound non-active oPL detected in sera of immunized ewes by western blotting indicated enhanced oPL production by the placenta following immunization. An increase (P<0.02) in serum oPL bioactivity, but not immunoreactivity, was observed in the immunized ewes in late gestation relative to control ewes. The average litter size was 1.83 and 1.32 lambs born per ewe lambing in the first and second experiments, respectively. Average birth weights of lambs born to the immunized ewes were higher (P<0.01) than for lambs born to control ewes by 10, 17 and 39% for those born as singles, twins and triplets, respectively. Immunized ewes produced 19 and 33% more milk (P<0.02) than the control ewes in the first 3.5 months of the first and second lactations, respectively. These findings do not suggest a role for oPL in maternal recognition of pregnancy, but they strongly suggest important roles for oPL in fetal growth and mammogenesis. Immunization of ewes against oPL may thus represent a novel practical technique for enhancing birth weights of lambs born to prolific sheep, as well as milk production by both dairy and mutton ewes.     

Passive immunization of rams (Ovis aries) against GnRH: effects on antibody titer,serum concentrations of testosterone,and sexual behavior

The effect of immunoneutralization of gonadotropin-releasing hormone (GnRH) on serum concentrations of testosterone and sexual behavior was evaluated in sexually mature male sheep. In Experiment 1, GnRH1 rams (n=16) were passively immunized against GnRH (300 ml antiserum), control rams were either passively immunized against keyhole limpet hemocyanin (KLH, n=15) or surgically castrated (Wethers1, n=4). Sexual performance of the rams was assessed weekly for 3 weeks before and 6 weeks after immunization, using ovarihystertomized ewes actively immunized against GnRH. Experiment 2 evaluated the effects of repeated immunization. Rams were immunized with two aliquots (400 and 300 ml, respectively) of anti-GnRH sera (GnRH, n=5) or normal sheep serum (NSS, n=4), 2 weeks apart. Surgically castrated animals were used as a second control group (Wethers2). Administration of anti-GnRH sera, but neither anti-KLH nor NSS sera, resulted in marked reduction (P<0.05) in serum concentrations of testosterone. Sexual behavior was not consistently affected by administration of one aliquot of anti-GnRH sera, however repeated immunizations resulted in more persistent reduction in serum concentrations of testosterone and more consistent suppression of sexual behavior.     

Early pregnancy diagnosis in goats by determination of pregnancy-associated glycoprotein concentrations in plasma samples

Different RIA systems available for measuring the concentrations of pregnancy-associated glycoproteins (PAGs) in dairy goats were compared in order to evaluate their accuracy in early pregnancy diagnosis. Plasma concentrations of PAGs were determined by 3 heterologous RIA systems with a bovine PAG standard and tracer in combination with antisera anti-ovine PAG (RIA 1), anti-caprine PAG55 + 62 (RIA 2), anti-caprine PAG55 + 59 (RIA 3), and by 2 homologous RIA systems that employed caprine PAG55 + 62 and caprine PAG55 + 59 and their specific antisera (RIAs 4 and 5, respectively). In all of the RIAs, the mean concentrations of PAGs were significantly higher (P < 0.01) in pregnant than in nonpregnant goats from Day 21 onwards after breeding. On Day 21, the accuracy rates of early pregnancy diagnoses were 56% (RIA 1), 96% (RIA 2), 99% (RIA 3), 95% (RIA 4) and 90% (RIA 5), whereas on Day 28 these rates were > 99% for RIAs 2, 3, 4 and 5. The RIAs for PAGs depend on proteins from the placenta being present in maternal plasma and require only a single sample of blood, to distinguish pregnant goats from those that fail to return to estrus for other reasons. The homologous and semi-heterologous assays are highly accurate as early as Day 21 of pregnancy.     

Multiple forms of Pregnancy-Associated Glycoproteins released in vitro by porcine chorion or placentomal and interplacentomal explants of wild and domestic ruminants

Characterization of the Pregnancy-Associated Glycoproteins (PAG) is important for studies of reproduction of various eutherian domestic, wild and endangered mammals. Distinct chorionic PAG genes are expressed in embryo-origin cells: pre-placental trophoblast (TR) and in placental trophectoderm (TRD) of various entherians. This study demonstrates in vitro production of the PAG proteins during long-term cultures of various chorionic explants: porcine TR or TRD, cotyledonary (CT) of European bison (Eb), and CT or intercotyledonary (intCT)-TRD of the cattle. Chorionic proteins isolated from media were analyzed by homologous or heterologous Western immunoblotting with anti-PAG sera, raised against cellular bovine or secretory porcine antigens. Used anti-PAG sera identified diverse molecular forms of released PAG proteins: 43-69 kDa for EbPAG proteins, 40-85 kDa for bovine PAG (bPAG), and 43-73 kDa for porcine PAG (pPAG). Immunoblotting revealed also that both CT and intCT-TRD explants secreted equivalent amounts of bPAG proteins. This useful system of in vitro protein production can provide native chorionic PAG proteins with placental unique carbohydrate chains. The PAG proteins are required as standard markers for diagnostic tests of pregnancy in domestic and wild mammals, in which seasonal reproductive processes are relatively difficult to control.     

Early pregnancy diagnosis in sheep by progesterone and pregnancy-associated glycoprotein tests

The aim of this study was to compare the accuracy of the progesterone (P4) and pregnancy associated glycoprotein (PAG) tests for determination of early pregnancy in sheep. Estrus was synchronized in 182 Awassi x Merino ewes and blood samples were collected at Days 0 (day of the insemination), 18, 22, 29, 36, and 50 after artificial insemination (AI). Plasma P4 concentrations at Days 0 and 18 were determined by double antibody radioimmunoassay, while PAG concentrations at Days 22, 29, 36 and 50 were determined by a heterologous, double-antibody radioimmunoassay (RIA) using the bovine PAG 67 kDa subunit as tracer and standard and rabbit antiserum raised against a mixture of caprine 55 and 59 kDa PAG subunits as the first antibody. The discriminatory value for diagnosis of pregnancy by the P4 and the PAG-RIA tests was > or = 1 ng/ml. Based on lambing data, the accuracy for diagnosing pregnant (sensitivity) and non-pregnant ewes (specificity) and predictivity of both tests were calculated. The sensitivity, specificity, positive and negative predictive values for P4 and PAG tests were 100, 95.4, 81.6, and 100% at Day 18 (P4) and 93.5, 100, 100 and 98.7% at Day 22 (PAG), respectively. For diagnosis of non-pregnant ewes the PAG test had significantly higher specificity than the P4 test (P < 0.05). It is concluded that ovine pregnancy can be reliably diagnosed at Day 22 after AI by using a heterologous radioimmunoassay of PAG.     

Combination of the ram effect with PGF2α estrous synchronization treatments in ewes during the breeding season

The role of the ram effect on the reproductive performance of ewes that have initiated estrous cycles following lambing in combination with synchronization of estrus using PGF(2α) was examined. A total of 1264 Corriedale × Merino ewes in the breeding season (March-April) were allocated to one of three treatments. The control group (PG2) of ewes (n=415) were in permanent direct contact with vasectomized rams throughout the experiment from 60 d prior to the administration of the first luteolytic dose of PGF(2α) which was followed by a second dose 13 d later (Day 0 of the experiment). Ewes assigned to the other two treatments remained isolated from rams until Day 0. In the second treatment, ewes (PG2RE; n=445) were administered PGF(2α) in the same manner and were joined with vasectomized rams at Day 0. Ewes allocated to the third treatment (PGRE; n=404) did not receive the second dose of PGF(2α) but were introduced to vasectomized rams on Day 0. Sexual receptivity, as indicated by tail-head marking, was recorded until d 11. More PG2RE ewes (407/445; 92%) were observed in estrus by Day 11 than occurred for PG2 ewes (353/415; 85%; P=0.003). The accumulated frequency of PG2RE ewes in estrus was greater than for PG2 ewes for each period from Day 3 (P<0.001) to Day 11 (P<0.01). The onset of estrus was earlier in PG2RE ewes (2.98±0.07 d) than for PG2 ewes (3.31±0.07 d; P<0.0001). In contrast, the total frequency of PGRE ewes observed in estrus by Day 11 (356/404; 88%) was similar to that observed for PG2 ewes. However, the trajectory of the accumulated frequency of the incidence of estrus was less for the PGRE ewes initially, particularly during the period of Days 3-6 of observation (P<0.0001). Consequently, onset of estrus was earlier in PG2 ewes (3.31±0.07 d) than for PGRE ewes (5.30±0.11 d; P<0.0001). It was concluded that the introduction of vasectomized rams simultaneously with the second administration of PGF(2α) advanced the onset of estrus and increased the number of ewes that responded. The introduction of rams 13 d after a single dose of PGF(2α) did not substitute for the second administration of PGF(2α).     

Effect of insulin on feed intake and reproductive performance of well-nourished nulliparous ewes

Eighty-four nulliparous ewes were used to examine the effect of short-term insulin treatment on feed intake and reproductive performance. Following estrus synchronization, ewes were observed for estrus (= Day 0) and were penned individually beginning on Day 7. Ewes were fed twice daily and feed intake was recorded. On Days 9 through 13, ewes were treated s.c. with 1 IU/kg BW insulin (n = 44) or an equivalent volume of saline (n = 40). On Day 14, ewes were placed with fertile rams and number of ewes in estrus (bred) was recorded. Thirty days post-breeding, ewes were checked for pregnancy via ultrasonography. Feed intake and percentage of ewes in estrus did not differ between saline- and insulin-treated ewes. Similarly, neither pregnancy rate (69 +/- 8.7% vs. 80 +/- 8.1%, respectively) nor lambing rate (61 +/- 8.9% vs. 78 +/- 8.4%, respectively) differed between treatments. The number of lambs born per ewe was, however influenced by a breed-group effect (P < 0.0002). Romanov ewes had more (P < 0.001) lambs than the other breed groups in the study. Therefore, treating well-nourished, nulliparous ewe lambs with insulin did not increase reproductive efficiency, possibly because the ewes were already at a maximal nutritional and/or reproductive state.     

Ultrasound fetal measurements and pregnancy associated glycoprotein secretion in early pregnancy in cattle recipients carrying somatic clones

Somatic cloning in the bovine species leads to high levels of fetal losses which occur throughout pregnancy. These losses are most often associated with fetal overgrowth, a syndrome known as large offspring syndrome (LOS), and excessive maternal plasma pregnancy serum protein 60 (PSP60), a protein similar to a pregnancy-associated glycoprotein of 67 kDa (PAG I67) produced by the bovine placenta. Predicting the outcome of pregnancies initiated from cloned embryos has become an important issue both to prevent potential harm to the mother because of excessive fetal size at birth and also to get a better understanding of the relationships between growth, differentiation and placental functions in developing cloned fetuses. Here, we report on a systematic analysis of fetal and placental development in the first trimester of pregnancy performed by ultrasonographic imaging and by measurement of the maternal concentrations of pregnancy associated glycoproteins (PAGS), using four different radioimmunoassays (RIA) (two homologous RIA systems with PSP60 and PAG I67; two heterologous RIA systems with PAG I67 as standard and tracer, and antisera anti-caprine PAGs). We showed that crown-rump length (CRL) in clones appeared smaller than controls at 35, 50 and 62 days (P<0.05). At 62 days of pregnancy, CRL in cloned fetuses that died before 90 days was smaller compared to the other cloned fetuses (P<0.05) whereas the width of the fetal sack and the biparietal diameter (BPD) was larger in fetuses that developed LOS in late gestation (P<0.05). Maternal PAGs concentrations were statistically different between controls and all clone recipients as early as Day 34, suggesting early abnormal placental glycoprotein synthesis for clone pregnancies regardless of pregnancy outcome. This work provides a practical, non-invasive tool to follow up clone pregnancies and suggests that primary growth retardation and abnormal placental function precedes excessive fetal and placental growth at later stages of pregnancy.     

Switching maternal dietary intake at the end of the first trimester has profound effects on placental development and fetal growth in adolescent ewes carrying singleton fetuses.

The aim was to investigate whether placental growth and hence pregnancy outcome could be altered by switching adolescent dams from a high to a moderate nutrient intake, and vice-versa, at the end of the first trimester. Embryos recovered from adult ewes inseminated by a single sire were transferred in singleton to peripubertal adolescents. After transfer, adolescent ewes were offered a high (H, n = 33) or moderate (M, n = 32) level of a diet calculated to promote rapid or moderate maternal growth rates, respectively. At Day 50 of gestation, half the ewes had their dietary intakes switched, yielding 4 treatment groups: HH, MM, HM, and MH. A subset of ewes were killed at Day 104 of gestation to determine maternal body composition in relation to growth of the products of conception. Maternal body composition measurements revealed that the higher live weight in the high-intake dams was predominantly due to an increase in body fat deposition, with a less pronounced increase in body protein. At Day 104, HH and MH groups (high intake during second trimester) compared with MM and HM groups (moderate intake during second trimester) had a lower (p < 0.002) total fetal cotyledon weight; but fetal weight, conformation, and individual organ weights were not significantly influenced by maternal dietary intake. In ewes delivering live young at term, a high plane of nutrition from the end of the first trimester (HH and MH groups) compared with moderate levels (MM and HM groups) was associated with a reduction in gestation length (p < 0.009), total placental weight (p < 0.002), total fetal cotyledon weight (p < 0.001), and mean fetal cotyledon weight per placenta (p < 0.001). Fetal cotyledon number was dependent on maternal dietary intake during the first trimester only and was lower (p < 0.007) in HH and HM ewes compared to MM and MH ewes. The inhibition of fetal cotyledon growth in HH and MH groups was associated with a major decrease (p < 0.001) in lamb birth weight at term relative to the MM and HM groups. Thus, reducing maternal dietary intake from a high to a moderate level at the end of the first trimester stimulates placental growth and enhances pregnancy outcome, and increasing maternal dietary intake at this time point has a deleterious effect on placental development and fetal growth.     

Effect of heat stress on ovine placental growth in early pregnancy.

Ditocous Dorset ewes were fed to predicted requirements and kept in environmental chambers at 21 degrees C (n = 6) or 40 degrees C (n = 5) between days 50 and 75 of gestation. Ewes were slaughtered and the pregnant uterus was dissected for measurement of conceptus weights and in vitro estimations of placental mitotic activity. Heat caused a 19% reduction in placental weight but did not affect fetal weight. Placental DNA and protein concentrations and protein/DNA were similar in both groups. Total placental DNA content was significantly reduced in the heated ewes, suggesting a reduction in cell number; however, DNA synthetic rate tended to be higher. These results are consistent with the hypothesis that fetal growth retardation in chronically heat-stressed ewes occurs in late pregnancy as a consequence of a primary reduction in placental growth in early gestation.     

Pattern of gonadotropin-releasing hormone (GnRH)-like stimuli sufficient to induce follicular growth and ovulation in ewes passively immunized against GnRH.

The pattern of GnRH-like stimuli capable of inducing follicular growth, ovulation, and luteal function was evaluated in ewes passively immunized against GnRH. The estrous cycles of 30 regularly cyclic sheep were synchronized using vaginal pessaries impregnated with a synthetic progestogen. Animals were passively immunized against GnRH (groups 2-5, n = 6) or the carrier protein, keyhole limpet hemocyanin (KLH; group 1, n = 6), at the time of pessary removal (PR). Circhoral delivery of saline (groups 1, 2, and 5) or low amplitude GnRH agonist (des-Gly10 GnRH ethylamide [100 ng/hourly pulse]; groups 3 and 4) was initiated at PR and continued for 3 (groups 4 and 5) or 12 days (groups 1-3). In groups 4 and 5, the amplitude of the GnRH-like stimulus was increased to 800 ng/hourly pulse (stimulus-shift) during the 24-h period beginning 72 h after PR. The amplitude of the hourly stimulus was adjusted to 100 ng/pulse 96 h after PR and continued at that level to Day 12. The endocrine changes associated with follicle growth and maturation (serum concentrations of estradiol [E2] above 10 pg/ml), ovulation (surge-like secretion of LH and FSH), and normal luteal function (serum concentrations of progesterone [P] above 2 ng/ml) were evident in ewes passively immunized against KLH (group 1). In this group, the preovulatory surge of gonadotropins was noted 48.7 +/- 1.2 h after PR. These endocrine events were blocked by passive immunization against GnRH (group 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Myometrial activity and plasma progesterone and oxytocin concentrations in cycling and early-pregnant ewes

Myometrial activity and plasma progesterone (P) and oxytocin (OT) were measured in early pregnant (n = 5) and cycling (n = 5) ewes. Electromyography (EMG) leads and jugular and inferior vena cava (IVC) catheters were surgically placed in ewes about 1 wk before data collection. When ewes returned to estrus, they were bred to either an intact or vasectomized ram. Continuous EMG data were collected, and blood samples were collected twice daily from day of estrus (Day 0) until Day 18. Ewes bred with an intact ram were checked surgically for pregnancy on Day 20. Computerized, quantitative analysis of EMG events showed no difference in signal from the right to left uterine horns, and no differences between pregnant and cycling ewes (p less than 0.05) until Days 14-18 when nonpregnant ewes returned to estrus and had increased EMG activity. The mean number of EMG events 180-900 s in length decreased in pregnant ewes, but this difference was not significant (p less than 0.05). Jugular plasma progesterone (P) levels confirmed corpus luteum (CL) formation in all ewes, and no differences in P between pregnant and nonpregnant ewes were measured until Days 14-18, when cycling ewes underwent luteolysis and pregnant ewes maintained CL. IVC plasma oxytocin concentrations were increased in pregnant ewes compared to concentrations in nonpregnant ewes on Days 5-13 (p less than 0.05), and the difference was largest at Day 6 (means +/- SEM pg/ml: pregnant = 68.7 +/- 13.9, nonpregnant = 30.9 +/- 19.9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Shearing during late pregnancy increases size at birth but does not alter placental endocrine responses in sheep

Shearing during the latter half of pregnancy is a common practice to improve flock health and productivity. Previous studies have demonstrated that shearing pregnant ewes at mid or late pregnancy is associated with an increase in lamb birth weight. In the present study, we used singleton Polypay × Dorset pregnant sheep, to investigate the potential roles of placental function and changes in maternal metabolism in underlying this increased birth weight response. Two groups were randomly established and blocked at enrollment by animal BW, body condition score and subcutaneous adipose tissue depth. The groups were shorn (SH; n = 18) or not (C; n = 20) at gestational day (GD) 107 ± 1 (mean ± SEM). Weekly maternal plasma samples were collected between shearing and birth, but only six samples were assayed for progesterone, pregnancy-associated glycoproteins (PAG1), glucose and non-esterified fatty acids (NEFAs). At birth, sex, birth weight, and newborn body mass index (BMI) were recorded. Maternal BW during mid- to late-pregnancy was similar between groups. Shearing resulted in increased lamb birth weight and BMI (P < 0.05) regardless of fetal sex but did not affect the maternal concentration of PAG1 or progesterone from GDs 100 to 142. After shearing (GD100) and up to lambing, shorn females had higher circulating glucose concentrations (P < 0.05), but not NEFA, compared to the control group. Maternal circulating PAG1, progesterone, glucose or NEFA concentration across pregnancy did not differ according to lamb sex. Across pregnancy, birth weight was positively associated with PAG1 (P < 0.001), but not with progesterone concentrations. In conclusion, weight and BMI at birth were higher in both sexes upon shearing in singleton pregnancies. Despite PAG1 being associated with birth weight, late-pregnancy shearing did not alter the placental endocrine response. Whether other placental factors are altered upon shearing and may influence the increase in birth weight and BMI remain to be investigated.