Validation and fine mapping of a QTL for ovulation rate on swine chromosome 3.

Ovulation rate (OR) is an important component of litter size, but mutation(s) in gene(s) underlying OR QTL have yet to be identified in pigs. Markers within an OR QTL on SSC3 were genotyped in three white composite lines selected for ten generations for increased OR or uterine capacity (UC), with one line being an unselected control. Numbers of corpora lutea (CL) and UC (number of fully formed fetuses) were collected at approximately 105 days of gestation, as well as ovary weight (OW), uterine length (UL) and uterine weight (UW) measurements at 160 d of age in generation 12 and 13 females from all three lines. Six microsatellites and ten single nucleotide polymorphisms (SNPs; 0-42 cM) were genotyped in pigs from all lines of generations 11 through 13. The allele frequencies of 24269.1, SW2429, 7907.2 and 7637.2 were different (P < 0.01) in the OR line compared to the control line. A significant (P < 0.05) association of CL with 24269.1 (additive effect 0.65 ± 0.32) was detected, and additive genotypic effects approached significance for markers at 28 through 35 cM (16963.2, 27514.1 and SWR1637). Haplotyping of 7637.2 and 16963.2 (31 through 32 cM) identified a significant additive association of haplotype 1 with CL (-0.62 ± 0.30). These markers were also associated with OW (24296.1 and SWR1637), UL (16963.2, 27514.1 and haplotypes of 7637.2/16963.2) and UW (haplotypes of 7637.2/16963.2). This study verifies an OR QTL on SSC3. However, based on the data, it was concluded that there may be two genes, at 13 through 18 cM and 28 through 35 cM, controlling OR on SSC3p.

Number of fetuses and conceptus growth throughout gestation in lines of pigs selected for ovulation rate or uterine capacity.

Selection for 11 generations in swine for ovulation rate (OR) or uterine capacity (UC) resulted in 19.6% greater prenatal survival at term in UC compared with OR. Our objective was to characterize the number of fetuses throughout gestation in each line, including an unselected control (CO) line. Five hundred ninety-three gilts produced over 4 farrowing seasons were subjected to unilateral-hysterectomy-ovariectomy at 160 d of age and mated within line at 280 d of age. Gilts were assigned within sire family to be slaughtered (+/- 2 d) at d 25, 45, 65, 85, or 105 of gestation. Ovulation rate and number of live and dead fetuses were recorded for each pregnant gilt (n = 402). Fetal and placental weights were also recorded. Ovulation rate of OR line gilts (18.0 +/- 0.3 ova) exceeded (P < 0.001) CO and UC lines (15.0 +/- 0.3 and 14.0 +/- 0.3 ova, respectively). Line and gestational age interacted to affect number of live fetuses (P < 0.001). Least squares means for CO were 10.1, 8.3, 7.2, 6.7, and 7.3 live fetuses for d 25, 45, 65, 85, and 105, respectively (average SE = 0.46 fetuses). Corresponding means for OR were 13.4, 8.3, 7.9, 6.5, and 6.7 live fetuses, respectively (average SE = 0.44 fetuses). Means for UC were 10.2, 9.0, 8.5, 7.5, and 8.0 live fetuses, respectively (average SE = 0.47 fetuses). In each line, number of live fetuses at d 25 was approximately 72% of ovulation rate. Mortality to d 45 was greatest in OR, intermediate in CO, and least in UC. Reductions in live fetuses continued to occur from d 45 to 105, but line differences at d 45 were essentially maintained to d 105. Number of live fetuses in gilts at d 114 was estimated from each of the survival curves and predicted values of 7.0, 5.9, and 7.8 per uterine horn for CO, OR, and UC lines, respectively. Selection for uterine capacity improved fetal survival primarily during the time period between d 25 and 45. Relative growth rate coefficients throughout gestation for placental tissue indicated a change in rank of the line means, implicating a relative later growth pattern of placental tissue in the UC line.

Comparison of meat composition from offspring of cloned and conventionally produced boars.

This study compares the meat composition of the offspring from boars produced by somatic cell nuclear transfer (n=4) to that of the offspring from conventionally produced boars (n=3). In total, 89 commercial gilts were artificially inseminated and 61 progressed to term and farrowed. All of the resulting piglets were housed and raised identically under standard commercial settings and slaughtered upon reaching market weight. Loin samples were taken from each slaughtered animal and shipped offsite for meat composition analysis. In total, loin samples from 404 animals (242 from offspring of clones and 162 from controls) were analyzed for 58 different parameters generating 14,036 and 9396 data points from offspring of clones and the controls, respectively. Values for controls were used to establish a range for each parameter. Ten percent was then added to the maximum and subtracted from the minimum of the control range, and all results within this range were considered clinically irrelevant. Of the 14,036 data points from the offspring of clones, only three points were found outside the clinically irrelevant range, two of which were within the range established by the USDA National Nutrient Database for Standard Reference, Release 18, 2005; website: (www.nal.usda.gov/fnic/foodcomp/search/). The only outlier was the presence of Eicosadienoic acid (C20:2) in one sample which is typically present in minute quantities in pork; no reference data were found regarding this fatty acid in the USDA National Nutrient Database. In conclusion, these data indicated that meat from the offspring of clones was not chemically different than meat from controls and therefore supported the case for the safety of meat from the offspring of clones.

A comparison of reproductive characteristics of boars generated by somatic cell nuclear transfer to highly related conventionally produced boars.

This study compares the reproductive performance of boars produced by somatic cell nuclear transfer versus conventional breeding. Two different genotypes were selected for comparison: terminal cross line 1 (TX1) and terminal cross line 2 (TX2). The boars selected for comparison from TX1 were three cloned boars, produced by somatic cell nuclear transfer and the conventionally produced progenitor of the clones. The boars selected for comparison from TX2 were a cloned boar produced by somatic cell nuclear transfer and two conventionally produced half sibling boars that were offspring of the progenitor of the clone. Semen from each boar was collected, extended, evaluated and shipped offsite. Upon arrival, the semen was reevaluated and utilized for artificial insemination of 89 commercial gilts, at least 12 gilts per boar, producing 625 piglets. Pregnancy rates were determined at day 30 and 110 of gestation; and farrowing rate and gestation length were recorded. Differences were observed in some of the semen characteristics analyzed with the clones usually possessing superior semen quality to the control, this likely being a result of age differences amongst the clones and controls. Additionally no differences were noted between the clones and controls (progenitor) or between individual boars within genetic line for pregnancy rates, gestation length or any of the litter parameters examined between the clones and controls. These data further support previous reports with limited numbers that the reproductive capabilities of cloned boars are equal to that of conventionally produced boars.

Lack of effect of metyrapone and exogenous cortisol on early porcine conceptus development.

A study was conducted to evaluate the influence of maternal cortisol on early conceptus development in pigs (Sus scrofa). The corticosteroid synthesis inhibitor metyrapone was injected daily during days 14-19 of pregnancy, without (n = 6) and with commensurate administration of cortisol (n = 6). Blood samples were taken via an indwelling jugular catheter on days 14 and 18, and conceptuses were harvested during surgery on day 20. Compared with vehicle-injected control dams (n = 7) plasma cortisol and aldosterone concentrations were decreased (P < 0.01) by 52 and 29%, respectively, by metyrapone treatment. Cortisol administration reversed decreases in plasma cortisol by day 18. There were no treatment-associated effects on conceptus survival or size. Nor were there treatment-associated effects on allantoic fluid volume or content. Trophodermal glucocorticoid receptor (GR) mRNA expression decreased by 34% (P < 0.05) in metyrapone-treated pigs, and was not further influenced by concomitant administration of cortisol, thereby suggesting an influence of aldosterone on GR mRNA expression. Also, when all pigs were considered, there were treatment-independent second-order polynomial regressions (P < 0.05) between maternal plasma cortisol concentrations and embryonic weight, allantoic size and allantoic glucose concentrations, and between plasma aldosterone concentrations and trophodermal GR mRNA expression. Such biphasic corticosteroid concentration versus tissue parameter curves are noteworthy, but difficult to interpret validly. They may suggest that an appropriate corticosteroid environment is necessary for optimal porcine embryonic development during this stage of gestation, but cannot overshadow the absence of treatment effects on the porcine embryonic measures evaluated.

Allelic variation in the secreted folate binding protein gene is associated with uterine capacity in swine.

Previous comparisons between the cDNA and gene sequences for secreted folate binding protein (sFBP) indicated a 12-bp insertion/deletion (ins/del) polymorphism in exon 1 and a SNP that altered (Ser-Arg) the protein AA sequence. The effect of the Ser-Arg SNP on reproductive traits was examined in three groups of Meishan-White European breed crossbred gilts. The gilts for all three groups were unilaterally hysterectomized-ovariectomized (UHO) at 100 d of age. Group 1 gilts (n = 77) were mated at estrus, slaughtered at d 105 of pregnancy, and a blood sample was collected from each fetus to determine fetal hematocrit. The number of corpora lutea and fetuses and the fetal and placental weights were recorded. Group 2 gilts (n = 46) were mated, the remaining uterine horn was flushed with 20 mL of saline on d 11 of pregnancy, conceptuses were counted, and flushings were measured for total sFBP. Gilts were allowed an estrous cycle to recover, mated again at estrus, slaughtered at 105 d of gestation, and the data as described for Group 1 were collected. Groups 1 and 2 gilts were genotyped for the Ser-Arg SNP. In Group 3, gilts (n = 70) and boars (n = 30) were genotyped for the Ser-Arg SNP before mating, and like genotypes were mated. Gilts were then treated as described for Group 2. The effect of the 12-bp ins/del on reproductive traits was examined in 407 white crossbred UHO gilts from a randomly selected control line and from lines selected for ovulation rate (OR) and uterine capacity (UC). Gilts were mated and slaughtered at 105 d of age, and the numbers of corpora lutea and live fetuses, and fetal and placental weights and fetal hematocrits were recorded. The 12-bp ins/del also was evaluated in 131 intact gilts from the OR selected line. These gilts were mated at approximately 250 d of age and farrowed. The numbers of fully formed and live piglets were recorded. A significant effect (P < 0.05) of the Ser-Arg SNP was detected on the number of embryos present on d 11 of pregnancy and on UC. The sFBP 12-bp ins/del was associated with UC (P < 0.01) and the number of CL (P < 0.05) in UHO gilts, but not with litter size in intact gilts from the OR line. Results suggest that the 12-bp ins/del polymorphism could be exploited to increase litter size in swine, provided that the negative effect of the polymorphism on OR is overcome.

Allelic variation in the erythropoietin receptor gene is associated with uterine capacity and litter size in swine.

A single nucleotide polymorphism (SNP; C vs. T) that creates an extra GATA-1 site (T allele) in intron 4 of the swine erythropoietin receptor (EPOR) gene was discovered and a genotyping assay for this SNP was developed. A total of 402 gilts from lines selected either at random (control), for ovulation rate (OR) or for uterine capacity (UC) for 11 generations were unilaterally hysterectomized-ovariectomized (UHO) at 160 days of age, mated at approximately 250 days of age and slaughtered at 105 days of pregnancy. Blood samples and spleens were collected from each foetus and the numbers of corpora lutea (CL) and live foetuses, the weights of each foetus and placenta, and each foetal haematocrit were recorded. In addition, intact gilts from the OR line or from a Yorkshire, Landrace, Duroc, crossbred line (BX) were mated and farrowed. At farrowing, the numbers of fully formed and live piglets were recorded for each litter. Genomic DNA was isolated for both the UHO and intact gilts, from foetuses from the UHO gilts that were heterozygous for the EPOR SNP, and from the boars from the BX line and were then used to determine EPOR SNP genotypes. Only CC and CT gilts were observed in the control, OR and UC selected lines. Presence of the EPOR T allele was associated (P < 0.05) with increased UC in these gilts. The number of heterozygous and homozygous foetuses did not differ within UHO litters, or did EPOR genotype influence foetal haematocrit. In intact gilts from the OR line, litter size was significantly associated (P < 0.05) with EPOR SNP genotype. Finally, results from intact gilts of the BX line, in which both the gilt and the boar genotypes were known, allowed an analysis to determine the effect of the gilt and/or the foetal genotype on litter size. This analysis indicated that the predicted foetal genotype (with gilt genotype as covariate) was associated with litter size (an increase of 2.6 +/- 1.0 piglets born alive predicted for homozygous T litters compared with homozygous C litters, P < 0.01) whereas the effect of the gilt genotype (adjusted for foetal genotype) on litter size was not significant. These results indicate that the EPOR SNP is associated with UC and litter size in two distinct populations and could be useful in increasing litter size in swine that are not limited in OR.

Molecular cloning and endometrial expression of porcine high density lipoprotein receptor SR-BI during the estrous cycle and early pregnancy.

During rapid development of the fetus, levels of high density lipoprotein (HDL) are elevated in pregnant women. The receptor for HDL, scavenger receptor class B type I (SR-BI), mediates selective cholesteryl ester uptake and is highly expressed in the human placenta. Because of the rapid growth of uterus during early pregnancy and differences in placentation between swine and humans, we hypothesized that SR-BI may be expressed in porcine endometrium to take up HDL cholesterol. The objectives of this study were to obtain the full coding region for porcine SR-BI, determine endometrial expression of SR-BI mRNA during the estrous cycle and early pregnancy, and map the gene. By iterative screening of a porcine expressed sequence tag library, we obtained the full coding region of SR-BI. Endometrial expression of SR-BI in White composite gilts (n = 3-4 each) was determined by Northern blotting on Days 10, 13, and 15 cyclic gilts and Days 10, 13, 15, 20, 30, and 40 pregnant gilts. In cyclic gilts, endometrial expression of SR-BI did not change between Days 10 and 13, but increased (P < 0.01) between Days 13 and 15. In pregnant gilts, endometrial expression of SR-BI increased (P < 0.01) between Days 10 and 13, remained elevated until Day 30, and decreased (P = 0.015) on Day 40. The SR-BI gene was mapped to 46.3 cM on chromosome 14. These results show that endometrial expression of SR-BI changes during the estrous cycle and early pregnancy, and suggest that SR-BI takes up HDL for endometrial development during early pregnancy.

Lack of an association between plasma follicle-stimulating hormone concentrations and ovarian weight in prepubertal gilts.

Selection for increased number of corpora lutea in gilts is associated with increased plasma FSH concentrations during pubertal development. In the current study, 270 gilts from a control (CO) line and a line selected for increased ovulation rate (OR) were unilaterally ovariectomized at 85 d of age, and this ovarian weight was related to FSH concentrations at 65, 75, and 85 d of age. Gilts were produced during two farrowing seasons, spring and fall, and the age at first estrus was monitored from 160 to 250 d. Plasma FSH was greater in OR than in CO gilts at 65 (P < 0.01) and 75 d (difference in spring greater than in fall, P < 0.01), but FSH at these ages was not correlated with ovarian weight at 85 d. At 85 d, FSH did not differ in gilts of these lines; however, FSH was negatively correlated (r = -0.27, P < 0.01) with ovarian weight. The proportion of gilts detected in estrus was less for spring-born CO gilts than for spring-born OR or for fall-born CO and OR gilts (78 vs. 92%, season x line, P < 0.02). The age at first estrus was similar in the two lines but was earlier (P < 0.01) for spring-born than for fall-born gilts (194 vs. 204 d). Concentrations of FSH at each of the ages examined were not correlated with the age at first estrus. These observations support the conclusion that selection for a greater number of corpora lutea produces a correlated increase in plasma FSH during early pubertal development. This increase in FSH most likely reflects differences in FSH synthesis and release and not differences in the stage of pubertal development.

Analysis of transcriptional control elements in the 5'-upstream region of ovine interferon-tau gene using feeder-independent caprine trophoblast cell line, HTS-1.

Interferon-tau (IFNtau) is a protein secreted from the embryonic trophectoderm of ruminant ungulates during peri-implantation period. This protein acts on the uterine endometrium, which indirectly maintains corpus luteum function, and is therefore considered essential for the process of maternal recognition of pregnancy. Transcriptional regulation of IFNtau genes had been examined using human choriocarcinoma cell lines, JEG-3 or JAR, however, molecular mechanisms by which cell and term specific IFNtau expression are regulated have not been elucidated. Recently, a feeder cell free-trophoblast cell line derived from Shiba-goat placenta, termed HTS-1, was established. In the present investigation, the 5'-upstream region of ovine IFNtau (oIFNtau) gene was analysed using this cell line, which would provide a more suitable system for studies of the ovine trophoblast specific gene than human choriocarcinoma cells. Variously modified 5'-upstream sequences of the oIFNtau gene fused to a luciferase reporter gene were transiently transfected into HTS-1 cells, and human JEG-3 cells were used as a control. These results and co-transfection with expression vectors revealed that Ets-2 binding site in the promoter region was important in HTS-1, whereas AP-1 that binds to the enhancer region was a major activator in JEG-3. By electrophoretic mobility shift assay, a nuclear protein from HTS-1 cells was confirmed to bind specifically to the Ets-2 site of oIFNtau promoter region. Differences in amounts of AP-1 and Ets-2 protein were demonstrated in nuclear extracts from HTS-1, JEG-3 and ovine conceptuses. Substantial differences on oIFNtau gene transcriptions found between caprine HTS-1 and human JEG-3 cells suggest that this cell line could be valuable in the elucidation of a molecular mechanism(s) by which oIFNtau gene expression is regulated in a cell specific manner.

Effect of progesterone, mifepristone, and estrogen treatment during early pregnancy on conceptus development and uterine capacity in Swine.

A series of experiments was performed to investigate the influence of progesterone at Days 2 and 3 of pregnancy on conceptus development and uterine capacity. In experiment 1, unilaterally hysterectomized-ovariectomized (UHO) white crossbred gilts were given no treatment, estradiol valerate (5 mg given on Days 11 and 12), or progesterone (200 mg/day on Days 2 and 3 after mating). On Day 105 of pregnancy, each fetus and its associated placenta were weighed, and the number of live and dead fetuses was recorded for each litter. Early progesterone treatment reduced (P < 0.05) litter size (a measure of uterine capacity in UHO gilts). In experiment 2, intact white crossbred gilts were mated, given no treatment or progesterone treatment on Days 2 and 3 of pregnancy, and farrowed. Progesterone treatment decreased (P < 0.05) pregnancy rates. In pregnant gilts, progesterone had no effect on the number of live or stillborn piglets at birth, and gestation length was decreased (P < 0.05). Progesterone treatment did not affect the number of large or small piglets. In experiment 3, intact gilts were mated at estrus and then received 1). no treatment or treatment with 2). 100 mg, 3). 200 mg, or 4). 400 mg mifepristone (also known as RU486) on Day 2 of pregnancy. On Day 11 of pregnancy, both uterine horns were flushed, the number and diameter of each conceptus was recorded, and the flushed material was assayed for total protein and acid phosphatase. The 400 mg mifepristone treatment decreased conceptus diameter (P < 0.05) and total protein (P = 0.06) in the uterine flushings. In experiment 4, UHO gilts were mated at estrus, injected with either corn oil (control) or mifepristone (400 mg) on Day 2 of pregnancy, and killed on Day 105 of pregnancy, and the number and weight of live fetuses and placentas was recorded. In contrast to the effect of progesterone treatment, mifepristone decreased uterine capacity by decreasing the number of small conceptuses. These data suggest that progesterone concentrations on Days 2 and 3 of pregnancy in swine influence the rate of conceptus development during early pregnancy and uterine capacity during later pregnancy.

The effect of breed and intrauterine crowding on fetal erythropoiesis on day 35 of gestation in swine.

In a previous report, it was suggested that intrauterine crowding impaired fetal erythropoiesis and that fetal erythropoiesis was accelerated in Meishan pigs during early pregnancy. Because these conclusions were based on limited numbers of observations, the present experiment was undertaken to provide a more extensive investigation of these phenomena. Intact white crossbred gilts, unilaterally hysterectomized-ovariectomized (UHO) white crossbred gilts, and intact Meishan gilts (n = 13 to 16 per group) were mated after at least one estrous cycle of normal duration (17 to 23 d). Gilts were laparotomized at d 35 of pregnancy, the uterine horns were exteriorized and opened near each fetus, and a blood sample was collected from each fetus. The uterine horn was then surgically removed, and each fetus and placenta was weighed. All fetal blood samples were measured for hematocrit, red blood cell number, and hemoglobin. Erythropoietin and the percentages of nucleated cells and reticulocytes were also measured in blood samples from the largest and smallest living fetus in each litter. Fetal hematocrits were not affected by treatment. Blood cell counts were greater (P < 0.01) in fetuses of Meishan gilts than in White crossbred intact or UHO gilts. Hemoglobin was less (P < 0.01) in fetuses of Meishan gilts than in fetuses of White crossbred intact or UHO gilts. The percentages of nucleated (immature) cells and reticulocytes were both less (P < 0.01) in fetuses of Meishan intact gilts. Erythropoietin was also lower (P < 0.01) in fetuses of Meishan gilts. As observed previously, fetal weight was correlated (r = 0.38; P < 0.01) with blood hemoglobin concentration. These results confirm that fetal erythropoiesis in Meishan gilts is accelerated compared with White crossbred gilts. These results are consistent with the hypothesis that faster blood cell development could be beneficial to fetal survival in swine.

11beta-hydroxysteroid dehydrogenase and glucocorticoid receptor messenger RNA expression in porcine placentae: effects of stage of gestation, breed, and uterine environment.

Glucocorticoids are known to influence many aspects of prenatal development. Three important regulators of glucocorticoid actions at the cellular level are the enzymes 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD-1), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD-2), and glucocorticoid receptors (GR). The present study was conducted to determine the presence of these regulators in porcine placentae during early gestation (Days 24-40; term = 114 days) and to examine the influence of breed and uterine environment. Three pig models differing in uterine environment as reflected by embryonic survival from Days 24 to 40 were used: intact white cross-bred gilts (WC-INT); white cross-bred gilts that had been unilaterally hysterectomized-ovariectomized before puberty (WC-UHO); and intact Meishan gilts (ME). Porcine-specific partial cDNAs for 11betaHSD-1 and 11betaHSD-2 and a cRNA for GRalpha were developed and used to produce 32P-labeled probes for Northern blot analyses. The 11betaHSD dehydrogenase activity was measured in vitro at saturating concentrations of substrate and coenzyme. At Day 24 of gestation, 11betaHSD-2 mRNA, dehydrogenase activity, and GR mRNA were present, but 11betaHSD-1 mRNA was absent. All three mRNAs and dehydrogenase activity increased (P < 0.01) by Day 40. On Day 30, placental 11betaHSD-2 mRNA was decreased (P = 0.03) by 47% in WC-UHO versus WC-INT. Placental 11betaHSD dehydrogenase activity was 2-fold greater (P < 0.01) in ME versus WC-INT on Day 24 of gestation. These results demonstrate, to our knowledge for the first time, the presence of 11betaHSD-1, 11betaHSD-2, and GR mRNA as well as 11betaHSD dehydrogenase activity in the porcine placenta during early pregnancy. Moreover, a role for glucocorticoids in porcine embryonic development is suggested.