B. A. Freking

Detection of transcriptional difference of porcine imprinted genes using different microarray platforms

BackgroundPresently, multiple options exist for conducting gene expression profiling studies in swine. In order to determine the performance of some of the existing microarrays, Affymetrix Porcine, Affymetrix Human U133+2.0, and the U.S. Pig Genome Coordination Program spotted glass oligonucleotide microarrays were compared for their reproducibility, coverage, platform independent and dependent sensitivity using fibroblast cell lines derived from control and parthenogenic porcine embryos.ResultsArray group correlations between technical replicates demonstrated comparable reproducibility in both Affymetrix arrays. Glass oligonucleotide arrays showed greater variability and, in addition, approximately 10% of probes had to be discarded due to slide printing defects. Probe level analysis of Affymetrix Human arrays revealed significant variability within probe sets due to the effects of cross-species hybridization. Affymetrix Porcine arrays identified the greatest number of differentially expressed genes amongst probes common to all arrays, a measure of platform sensitivity. Affymetrix Porcine arrays also identified the greatest number of differentially expressed known imprinted genes using all probes on each array, an ad hoc measure of realistic performance for this particular experiment.ConclusionWe conclude that of the platforms currently available and tested, the Affymetrix Porcine array is the most sensitive and reproducible microarray for swine genomic studies.

Effect of empty uterine space on birth intervals and fetal and placental development in pigs

A substantial loss of embryos occurs between Days 30 and 40 of pregnancy in the pig under crowded intrauterine conditions, but it is not clear whether this loss affects the growth of adjacent conceptuses. Birth intervals are known to increase with decreasing litter size, but the factors responsible are unknown. Two possibilities are that increased birth weight associated with reduced litter size and the empty uterine space and resulting constricted uterine regions that occur in pigs with small litters may impair piglet delivery. To address these, pregnant gilts were laparotomized on Day 35 of pregnancy and one or two fetuses were manually crushed through the uterine wall on the ovarian or cervical end of each uterine horn to create an empty uterine space behind or in front of the litter of piglets, respectively, in relation to the route of delivery from the uterus. A subset of gilts was slaughtered at 105 days of gestation to confirm that the empty uterine spaces were successfully created and to determine their effects on placental and fetal weights of adjacent conceptuses. At slaughter, the lengths of all externally visible empty constricted regions of the uterus were measured. The uterine horns were opened and the lengths of each placenta were measured from the umbilicus toward the ovary and toward the cervix to assess whether placentas developed symmetrically, and then each fetus and placenta was weighed. Fetal crushing successfully created constricted empty uterine regions on the ovarian and cervical ends of the uterine horns. Ovarian-side placental lengths were greater than cervical-side for conceptuses adjacent to fetuses crushed on the ovarian end of the horn. Cervical-side placental lengths were greater than ovarian-side for conceptuses adjacent to fetuses crushed on the cervical end. Both placental and fetal weights were greater (10% and 6%, respectively, P<0.05) for conceptuses adjacent to crushed fetuses compared to nonadjacent conceptuses. Remaining gilts were farrowed to determine the effect of litter size, average birth weights, and treatment on birth intervals of piglets, which were monitored using 24-h video surveillance. The negative association between number of piglets born alive and average birth interval was confirmed and was not explained by litter size-induced reduction in litter average birth weights. Birth intervals and stillbirth rate did not differ between cervically- and ovarian-treated gilts. These results indicate that conceptus loss on Day 35 of gestation can benefit the growth of adjacent placentas and fetuses, but the benefit is small. Increased average birth weight and the presence of empty uterine space that occurs when litter size is reduced does not fully explain the effect of litter size on birth intervals.

Selection for uterine capacity improves lifetime productivity of sows

Selection for 11 generations for uterine capacity (UC) increased litter size in gilts by 1.6 more fully formed pigs at birth compared to an unselected control line (CO) despite averaging one less ovulation. The objective of the present study was to quantify line by parity interactions and characterize litter performance traits of sows in each line at later parities. Gilts farrowed in contemporary groups of 19 litters and were maintained through four parities if successfully mated in that contemporary group. A total of 243 litters and 2639 piglets were analyzed. Fixed effects of farrowing group, line, parity (1-4), and two-way interactions involving line were fitted. Sire (n=57) of the sow within farrowing group and line was fitted as a random effect. No significant line by parity interactions were observed. Parity effects were detected (P<0.01) for individual piglet birth weight, pre-weaning gain, and weaning weight. Parity effects were also detected (P<0.05) for total number born, average and total litter birth weight, and average and total litter weaning weight. Selection line differences for litter traits were detected (P<0.05) for number stillborn piglets and approached significance (P=0.06) for number of piglets weaned. Retention of sows in the herd was greater (P<0.05) with an average of 2.33 parities for the UC line females compared to 1.87 parities for the CO line. This resulted in favorable cumulative lifetime productivity of the UC line for total number of piglets born, number of piglets born alive, litter birth weight, number of piglets weaned and litter weaning weight.

Differences in X-Chromosome Transcriptional Activity and Cholesterol Metabolism between Placentae from Swine Breeds from Asian and Western Origins

To gain insight into differences in placental physiology between two swine breeds noted for their dissimilar reproductive performance, that is, the Chinese Meishan and white composite (WC), we examined gene expression profiles of placental tissues collected at 25, 45, 65, 85, and 105 days of gestation by microarrays. Using a linear mixed model, a total of 1,595 differentially expressed genes were identified between the two pig breeds using a false-discovery rate q-value ≤0.05. Among these genes, we identified breed-specific isoforms of XIST, a long non-coding RNA responsible X-chromosome dosage compensation in females. Additionally, we explored the interaction of placental gene expression and chromosomal location by DIGMAP and identified three Sus scrofa X chromosomal bands (Xq13, Xq21, Xp11) that represent transcriptionally active clusters that differ between Meishan and WC during placental development. Also, pathway analysis identified fundamental breed differences in placental cholesterol trafficking and its synthesis. Direct measurement of cholesterol confirmed that the cholesterol content was significantly higher in the Meishan versus WC placentae. Taken together, this work identifies key metabolic pathways that differ in the placentae of two swine breeds noted for differences in reproductive prolificacy.

Measures of the ovaries and uterus during development of gilts selected for differences in uterine capacity.

Direct selection for uterine capacity (UC) increased the number of live pigs born. A method to indirectly estimate UC in developing gilts is needed for this trait to be incorporated into commercial selection strategies. We tested the hypothesis that selection for UC alters phenotypic characteristics of the reproductive tract of prepubertal gilts and that these changes could be estimated in live animals using transrectal ultrasound (TRU). Gilts from lines selected for UC and a randomly selected control (CO) line were submitted for TRU at 130, 150, or 170 d of age and harvested 24 h later (n = 10 gilts/line per age). Diameter of the uterine horn was measured (2 to 4 measurements per animal) at TRU. At harvest, measurements of the ovary (height, width, length, weight, and number of visible follicles >1 mm) and uterine horn (weight, length, diameter, and endometrial diameter) were taken. There was no line × age interaction for any of the traits. All ovarian traits increased (P < 0.03) with age. Weight and length of the uterine horn was not different at 130 or 150 d but were increased (P < 0.01) at 170 d of age. Diameter of the uterine horn tended (P = 0.06) to be increased at 170 d compared with 130 or 150 d, but age did not affect (P = 0.38) endometrial diameter. Ovarian weight and width were increased (P < 0.01) and ovarian length and height tended (P = 0.07) to be greater for UC than CO gilts, but the number of visible follicles did not differ. The UC gilts had increased (P < 0.02) weight of uterine horns, which tended (P = 0.09) to be longer than in the CO gilts. Diameter of the uterine horn and the endometrium were greater (P < 0.01) for UC gilts than for CO gilts. Uterine horn diameter measured with TRU was not affected by age or line and was not highly correlated with any of the measured traits. Selection for increased UC results in larger ovaries and uterine horns, but TRU was not useful for estimating these traits in gilts of the age studied.

Litter-of-origin trait effects on gilt development

The preweaning litter environment of gilts can affect subsequent development. In a recent experiment designed to test the effects of diet on gilt development, litter-of-origin traits including individual birth weights, immunocrits (a measure of colostrum intake), sow parity, number weaned, and individual weaning weights were collected for approximately 1,200 gilts that were progeny of approximately 300 sows. Subsequently, BW, LM area, and backfat were measured at 100 d of age and at 28-d intervals until slaughter (260 d of age). From 160 d of age to slaughter, gilts were observed daily for estrus. At slaughter, the reproductive tract and 1 mammary gland were recovered. The reproductive tract was classified as cyclic or prepubertal; the number of corpora lutea was counted. Uterine horn lengths and ovarian dimensions were measured. Uterus and ovary samples from every 10th gilt were prepared for histological evaluation of uterine gland development and follicle counts, respectively. Mammary gland tissue protein and fat were assayed. Day of the estrous cycle at slaughter was calculated using the first day of the most recent standing estrus (d 0) recorded previous to slaughter. Each gilt development trait was analyzed for association with each litter-of-origin trait, after adjusting for dietary treatment effects. Uterine length, ovarian dimensions, mammary gland protein and fat, and uterine gland development were also adjusted for day of the estrous cycle at slaughter. All litter-of-origin traits were associated ( < 0.05) with growth traits. Top-down (backward elimination) multiple regression analysis indicated that BW and LM accretion in gilts was positively associated with immunocrit ( < 0.01), birth weight ( < 0.01), preweaning growth rate ( < 0.01), and parity ( < 0.01). Backfat accretion was positively associated with preweaning growth rate ( < 0.01), number weaned ( < 0.05), and parity ( < 0.05). Age at puberty was associated with birth weight (positive; < 0.01) and preweaning growth rate (negative; < 0.01). Total uterine length was positively associated with only birth weights ( < 0.05). Mammary gland protein was negatively associated with preweaning growth ( < 0.01). Mammary gland fat was positively associated with birth weight and number of piglets weaned ( > 0.05). These results indicate that colostrum consumption, birth weights, preweaning growth rate, number weaned, and parity are associated with gilt development traits during later life.

Relationship of neuropeptide FF receptors with pubertal maturation of gilts

Abstract Mechanisms governing the timing of puberty in pigs are poorly understood. A genome-wide association study for age at first estrus in pigs identified candidate genes including neuropeptide FF receptor 2 (NPFFR2), which is a putative receptor for RFamide-related peptides (RFRP). RFRP has been shown to negatively regulate secretion of reproductive hormones from hypothalamic and pituitary tissue of pigs in culture. Here, the porcine NPFFR2 gene was further screened and four potentially functional variants were identified to be associated with age at first estrus in pigs (1,288 gilts). The RFRP neurons in the porcine hypothalamus were localized in the paraventricular and dorsomedial nuclei with RFRP fibers in the lateral hypothalamic area. There were marked changes in expression of NPFF receptors in the anterior pituitary gland and hypothalamus of gilts beginning with the peripubertal period. The hypothesis that NPFF receptor function is related to secretion of luteinizing hormone (LH) in gilts was tested with various NPFF receptor ligands. The NPFF receptor antagonist RF9 stimulated a pulse-like release of LH in prepubertal gilts. The putative NPFF receptor agonist RFRP3modestly suppressed LH pulses in ovariectomized (OVX) prepubertal gilts. A porcine-specific RFRP2 failed to have an effect on LH secretion in OVX prepubertal gilts despite its high degree of homology to avian gonadotropin-inhibitory hormone. Results indicate that an RFRP system is present in the pig and that NPFFR2 is important for pubertal onset in gilts. It is not clear if this regulation involves major control of LH secretion or another unknown mechanism. Summary Sentence Neuropeptide FF receptors play a role in attainment of puberty in pigs.

The relationship of plasma urea nitrogen with growth traits and age at first estrus in gilts

Gilts that reach puberty at an earlier age with more backfat have greater lifetime productivity. Increased growth rates generally promote earlier age at first estrus; however, an association of age at first estrus with discrete measures of body fatness remains controversial. We tested the hypothesis that metabolic state as determined by concentrations of plasma urea nitrogen (PUN), which reflect lean tissue growth, were correlated with age at first estrus. Blood samples were collected from gilts (n = 337) at 102, 123, and 145 d of age during development. Concentrations of albumin, creatinine, glucose, and PUN were determined. Body weight and backfat thickness were determined at each time point. From 130 to 240 d of age, gilts were monitored for first pubertal estrus. Concentrations of creatinine increased whereas concentrations of glucose decreased with increasing age (P < 0.0001). Concentrations of albumin and PUN remained relatively stable throughout development. Average daily BW gain (r = 0.22) and change in backfat thickness (r = 0.29) had a positive phenotypic correlation (P < 0.0001) with PUN at 145 d of age. Concentrations of PUN at 102 and 123 d of age were not phenotypically correlated with pubertal age, but there was a moderately negative phenotypic correlation (r = -0.22; P < 0.0001) of PUN at 145 d of age with age at first estrus along with a negative genetic correlation (r = -0.42). The relationship of PUN with age at first estrus shifted from liner to quadratic with advancing age. These data demonstrate that near the age at which gilts are selected for entry into the breeding unit, those with greater PUN have increased BW and backfat thickness and display pubertal estrus earlier but that PUN does not account for additional variation in age at first estrus beyond growth rate or backfat. It is concluded that PUN can be used to select gilts with increased efficiency of nutrient use without negatively impacting pubertal development.

Plasma concentrations of acyl-ghrelin are associated with average daily gain and feeding behavior in grow-finish pigs.

The objectives of this study were to determine the effect of sex, sire line, and litter size on concentrations of acyl-ghrelin and total ghrelin in plasma of grow-finish pigs and to understand the relationship of plasma concentrations of ghrelin with feeding behavior, average daily gain (ADG), and back fat in grow-finish swine. Yorkshire-Landrace crossbred dams were inseminated with semen from Yorkshire, Landrace, or Duroc sires. Within 24 h of birth, pigs were cross-fostered into litter sizes of normal (N; >12 pigs/litter) or small (S; ≤ 9 pigs/litter). At 8 wk of age, pigs (n = 240) were blocked by sire breed, sex, and litter size and assigned to pens (n = 6) containing commercial feeders modified with a system to monitor feeding behavior. Total time eating, number of daily meals, and duration of meals were recorded for each individual pig. Body weight was recorded every 4 wk. Back fat and loin eye area were recorded at the conclusion of the 12-wk feeding study. A blood sample was collected at week 7 of the study to quantify concentrations of acyl- and total ghrelin in plasma. Pigs from small litters weighed more (P < 0.05) and tended (P = 0.07) to be fatter than pigs from normal litters. Postnatal litter size did not affect ADG, feeding behavior, or concentrations of ghrelin in plasma during the grow-finish phase. Barrows spent more time eating (P < 0.001) than gilts, but the number of meals and concentrations of ghrelin did not differ with sex of the pig. Pigs from Duroc and Yorkshire sires had lesser (P < 0.0001) concentrations of acyl-ghrelin than pigs from Landrace sires, but plasma concentrations of total ghrelin were not affected by sire breed. Concentrations of acyl-ghrelin were positively correlated with the number of meals and negatively correlated with meal length and ADG (P < 0.05). A larger number of short-duration meals may indicate that pigs with greater concentrations of acyl-ghrelin consumed less total feed, which likely explains why they were leaner and grew more slowly. Acyl-ghrelin is involved in regulating feeding behavior in pigs, and measuring acyl-ghrelin is important when trying to understand the role of this hormone in swine physiology.

Contributions of the maternal uterine environment and piglet genotype on weaning survivability potential: II. Piglet growth, lactation performance, milk composition, and piglet blood profiles during lactation following reciprocal embryo transfers between Meishan and White crossbred gilts.

Crossbreeding studies between Meishan (MS) and Large White (LW) pigs have illustrated that increased piglet growth before weaning is attributed to the maternal genotype of LW dams. The objective of this study was to determine the contributions of the maternal uterine environment (MUE), piglet genotype (PigG), piglet age (PA), and their interactions on piglet growth, lactation performance, milk composition, and piglet blood profiles during lactation following reciprocal embryo transfers between MS and White crossbred (WC) gilts. Twenty-five successful pregnancies were generated by embryo transfer in 2 farrowing years representing all MUE × PigG combinations: MS × MS (n = 4 litters), MS × WC (n = 7 litters), WC × MS (n = 7 litters), and WC × WC (n = 7 litters). At d 1 and 10 and at weaning, piglets (n = 147, n = 96, and n = 94, respectively) were weighed and blood samples were collected and measured for hematocrit, hemoglobin, glucose, nitrogen, NEFA, albumin, lactate, and cortisol. In addition, sows were manually milked from a medial mammary gland to determine milk composition. All data were analyzed by ANOVA using MIXED model procedures with the fixed effects of MUE, PigG, PA, and their interactions. Piglet weight was greater (P < 0.001) in piglets from WC dams compared to MS dams at d 10 and weaning but not at d 1. In addition, ADG were greater (P < 0.05) from piglets from WC dams compared to MS dams throughout lactation. However, milk composition was greater (P < 0.05) for GE and fat content from MS dams compared to WC dams, illustrating differences in milk quality between the breeds. There were significant MUE × PigG × PA interactions for hematocrit and hemoglobin levels in which greater (P < 0.001) levels were observed in MS piglets, irrespective of MUE, at d 1 of lactation and in MS piglets from MS dams at d 10 of lactation. Blood glucose was greater (P = 0.01) at d 1 in piglets from WC dams regardless of PigG but, at weaning, glucose was greater (P = 0.01) in WC piglets regardless of MUE. Serum NEFA levels were greater (P = 0.02) in piglets from MS dams throughout the lactation period. This study demonstrated that WC dams were superior to MS dams for piglet growth during lactation, in agreement with previous crossbreeding studies. However, blood components measured displayed complex interactions between the piglet and maternal breed, which signify possible mechanisms for improved preweaning survivability but slower lactational growth of MS piglets.