J. R. Miles

MicroRNA transcriptome profiles during swine skeletal muscle development

BackgroundMicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult.ResultsTwelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus) and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate.ConclusionThese data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.

MicroRNA expression profile in bovine cumulus-oocyte complexes: Possible role of let-7 and miR-106a in the development of bovine oocytes

The objectives of this study included: (1) identify the expression of miRNAs specific to bovine cumulus-oocyte complexes (COCs) during late oogenesis, (2) characterize the expression of candidate miRNAs as well as some miRNA processing genes, and (3) computationally identify and characterize the expression of target mRNAs for candidate miRNAs. Small RNAs in the 16-27 bp range were isolated from pooled COCs aspirated from 1- to 10-mm follicles of beef cattle ovaries and used to construct a cDNA library. A total 1798 putative miRNA sequences from the cDNA library of small RNA were compared to known miRNAs. Sixty-four miRNA clusters matched previously reported sequences in the miRBase database and 5 miRNA clusters had not been reported. TaqMan miRNA assays were used to confirm the expression of let-7b, let-7i, and miR-106a from independent collections of COCs. Real-time PCR assays were used to characterize expression of miRNA processing genes and target mRNAs (MYC and WEE1A) for the candidate miRNAs from independent collections of COCs. Expression data were analyzed using general linear model procedures for analysis of variance. The expression of let-7b and let-7i were not different between the cellular populations from various sized follicles. However, miR-106a expression was greater (P<0.01) in oocytes compared with COCs and granulosa cells. Furthermore, all the miRNA processing genes have greater expression (P<0.001) in oocytes compared with COCs and granulosa cells. The expression of potential target mRNAs for let-7 and let-7i (i.e., MYC), and miR-106a (i.e., WEE1A) were decreased (P<0.05) in oocytes compared with COCs and granulosa cells. These results demonstrate specific miRNAs within bovine COCs during late oogenesis and provide some evidence that miRNAs may play a role regulating maternal mRNAs in bovine oocytes.

A simple novel measure of passive transfer of maternal immunoglobulin is predictive of preweaning mortality in piglets

Two factors that contribute to preweaning mortality in piglets are the initiation of lactation by sows and their ability to nurse their piglets. The objective of this study was to determine if the quantification of the transfer of immunoglobulins (Igs) from sow to piglet could act as a measure of these sow factors in terms of their influence on preweaning mortality. To measure passive transfer, a simple, rapid Ig immunocrit method was developed. For validation, the smallest piglets from 204 gilts were sacrificed on day 1 after birth and blood was collected. Piglet serum Ig concentrations were measured three ways: (1) by protein A sepharose precipitation, SDS-PAGE, and densitometry of the IgG heavy chain; (2) by precipitation of Ig with (NH(4))(2)SO(4) followed by spectrophotometric measurement; and (3) by precipitation of Ig with (NH(4))(2)SO(4) and measurement of the ratio of precipitate to sample volume using a hematocrit microcapillary (the Ig immunocrit method). Results from the (NH(4))(2)SO(4) methods correlated (r=0.86) with those obtained using SDS-PAGE. Day 1 weights and immunocrit ratios and preweaning survival data were then collected from every piglet from first (n=90), and second (n=145) parity sows. Bodyweight and immunocrit ratios accounted for 4.8% and 16.7% (P<0.01) of the variation in preweaning mortality, respectively. Litter average immunocrit ratios were not correlated with preweaning mortality. In conclusion, the Ig immunocrit method is a simple, rapid measure of passive transfer from sow to piglet, and is useful in assessing the initiation of colostrum and the nursing ability of sows, and the effect of these events on preweaning piglet mortality.

Adipose and Muscle Tissue Gene Expression of Two Genes (NCAPG and LCORL) Located in a Chromosomal Region Associated with Cattle Feed Intake and Gain

A region on bovine chromosome 6 has been implicated in cattle birth weight, growth, and length. Non-SMC conodensin I complex subunit G (NCAPG) and ligand dependent nuclear receptor corepressor-like protein (LCORL) are positional candidate genes within this region. Previously identified genetic markers in both genes were associated with average daily gain (ADG) and average daily feed intake (ADFI) in a crossbred population of beef steers. These markers were also associated with hot carcass weight, ribeye area and adjusted fat thickness suggesting that they may have a role in lean muscle growth and/or fat deposition. The purpose of this study was to determine whether the transcript abundance of either of these genes in cattle adipose and muscle tissue was associated with variation in feed intake and average daily gain phenotypes. Transcript abundance for NCAPG and LCORL in adipose and muscle tissue was measured in heifers (adipose only), cows and steers using real-time polymerase chain reaction. In the adipose tissue from cows and heifers, a negative correlation between LCORL transcript abundance and ADFI were detected (P = 0.05). In the muscle tissue from cows, transcript abundance of NCAPG was associated with ADG (r = 0.26; P = 0.009). A positive correlation between LCORL transcript abundance from muscle tissue of steers and ADFI was detected (P = 0.04). LCORL protein levels in the muscle of steers were investigated and were associated with ADFI (P = 0.01). These data support our earlier genetic associations with ADFI and ADG within this region and represent the potential for biological activity of these genes in the muscle and adipose tissues of beef cattle; however, they also suggest that sex, age and/or nutrition-specific interactions may affect the expression of NCAPG and LCORL in these tissues.

Comparative Transcriptome Analysis of In Vivo- and In Vitro-Produced Porcine Blastocysts by Small Amplified RNA-Serial Analysis of Gene Expression (SAR-SAGE)

Production of embryos in vitro has enormous potential for research and commercial applications. Unfortunately, in vitro production of porcine embryos is extremely inefficient. Despite the characterization of distinct phenotypes, little is known about the molecular mechanisms and altered physiological processes of in vitro‐produced embryos. The objective of this study was to compare global gene expression patterns from in vivo‐ (IVO) and in vitro‐produced (IVP) porcine embryos using small amplified RNA‐serial analysis of gene expression (SAR‐SAGE). Whole‐cell RNA from pools of Day 6 IVO and IVP blastocysts was used to construct SAR‐SAGE libraries. Sequence analysis of the IVO and IVP libraries yielded 98,771 and 98,408 tags, respectively. A total of 20,029 and 23,453 putative transcripts were detected in the IVO and IVP libraries, respectively. Statistical analyses of SAGE tag frequencies between the IVO and IVP libraries indicated that 938 and 193 tags were differentially expressed at a P < 0.05 and P < 0.001 level of significance, respectively, suggesting significant deviations in transcriptome profiles from IVO and IVP embryos. Categorization of differentially expressed transcripts into functional groupings indicated a significant deviation in gene expression from IVP blastocysts compared with IVO blastocysts for a number of biological processes including cellular metabolism, organization, and response to stress. Real‐time PCR confirmed differential expression for several transcripts from independent IVO and IVP blastocysts. These results demonstrate compromised gene expression in IVP blastocysts compared with IVO blastocysts for a number of biological processes, particularly processes involved in mitochondrial function; thereby providing potential target pathways for improvement of IVP methods. Mol. Reprod. Dev. 75: 976–988, 2007.

Proportion of the litter farrowed, litter size, and progesterone and estradiol effects on piglet birth intervals and stillbirths.

Stillbirth in swine ranges from 2 to 9%, resulting in a significant loss of piglets. Previous studies clearly indicate a relationship between prolonged birth intervals and stillbirth, but factors influencing birth intervals are not fully known. To characterize birth intervals and stillbirth, farrowing was recorded during three farrowing seasons. Blood samples were collected on d 110 and d 113 of gestation, and were assayed for progesterone and estrogen. Relationships between estrumate (cloprostenol sodium, an analogue of prostaglandin F(2alpha)) usage, litter size, proportion of the litter farrowed, progesterone and estrogen concentrations, birth intervals, and stillbirth were analyzed using regression analysis. A clear relationship between birth intervals and stillbirth was observed. Stillbirth rate was unaffected by birth intervals of <1 h, and increased (P < 0.01) for birth intervals >1 h. A significant negative association between litter size and birth intervals was observed (P < 0.01). Birth intervals were unaffected by proportion of the litter farrowed until the last piglet in the litter, whose birth interval increased dramatically (1.5-fold; P < 0.01). Stillbirth rates increased as proportion of the litter farrowed increased, and a dramatic increase in stillbirth occurred for the last piglet in the litter. Neither d 110 nor 113 plasma progesterone concentrations were associated with litter size, birth intervals, or stillbirth rates. Curvilinear relationships were present between d 110 or 113 plasma estradiol concentrations and litter size. However, neither d 110 nor 113 estradiol concentrations were associated with birth intervals or stillbirth rates. These results indicate that (1) birth intervals greater than 1 h are associated with increased stillbirth; (2) larger litter size reduces birth intervals; (3) the last piglet in the litter has both a prolonged birth interval and increased risk of stillbirth; (4) plasma progesterone before farrowing does not influence birth intervals or stillbirth; and (5) plasma estradiol does not influence birth interval or stillbirth, despite a positive relationship between litter size and plasma estradiol. An understanding of the effects of litter size and proportion of the litter farrowed on birth intervals might be exploited to decrease stillbirth in piglets.

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.

Molecular cloning and characterisation of heparanase mRNA in the porcine placenta throughout gestation

Heparanase (HPSE) is an endoglycosidase that specifically degrades heparan sulfate, which is an abundant glycosaminoglycan of the pig placenta. The aim of the present study was to clone cDNA encoding porcine HPSE and characterise the expression level and localisation of HPSE mRNA in porcine placentas throughout gestation. Placental tissues were collected from litters on Days 25, 45, 65, 85 and 105 of gestation. Three transcript variants similar to HPSE were identified in the pig placenta. In addition, the HPSE gene was mapped to pig chromosome 8 in close proximity to quantitative trait loci for litter size and prenatal survival. Real-time polymerase chain reaction and in situ hybridisation were used to characterise the expression of two HPSE variants, namely HPSE v1 and v2, in the pig placenta throughout gestation. The expression of HPSE v1 and v2 was elevated (P < 0.01) in placentas during very early gestation (Day 25) as well as during late gestation (Days 85 and 105). Finally, HPSE v1 and v2 mRNA were localised to the cuboidal trophoblast cells of the folded bilayer located nearest to the maternal endometrium. These findings illustrate that HPSE likely plays a role in the development and modification of the pig placenta, which has implications for litter size and prenatal survival.

Relationships between day one piglet serum immunoglobulin immunocrit and subsequent growth, puberty attainment, litter size, and lactation performance

Colostrum affects gut and uterine gland development in the neonatal piglet, suggesting that subsequent growth and reproductive performance may be affected. Measuring immunoglobulin in piglet serum using the immunoglobulin immunocrit on Day 1 of age provides a simple, inexpensive indication of the amount of colostrum acquired by the piglet in the first day of life. Relationships between serum immunoglobulin immunocrit measures and subsequent growth rates, age at puberty, incidence of puberty failure, litter size, and lactation performance were examined in pigs born and subsequently farrowing between 2009 and 2013. Immunoglobulin immunocrit measures were collected on 16,762 piglets on Day 1 of age. Of these piglets, BW measurements were available from 15,324 (7,684 males and 7,640 females) piglets at a range of ages from weaning to 200 d of age, allowing an assessment of growth rates. Age at puberty was recorded from a subset of 2,857 of the females after observing them for estrous behavior from approximately 170 to 250 d of age. To examine relationships between d 1 immunocrit and puberty failure, gilts with immunocrit measures that failed to reach puberty (n = 119) were matched with littermate gilts with immunocrit measures that achieved puberty (n = 167). Similarly, number born alive was collected on a subset (n = 799) of females from first to fourth parities for which d 1 immunocrits were measured on them as neonates. Finally, d 1 immunocrit effect on adult lactational competence was assessed by measuring litter average (offspring of 440 females) and litter average piglet preweaning growth rate (offspring of 774 females) in females where d 1 immunocrits were available from them as neonates. Results indicated that low d 1 immunocrits were subsequently associated with reduced growth (P < 0.01), increased age at puberty (P < 0.01), reduced number born alive (P < 0.05), reduced litter average immunocrit (P < 0.05), and reduced litter average preweaning growth rate during lactation (P < 0.05). This suggests that management efforts to improve the amount of colostrum ingested by neonatal piglets would result in beneficial changes in production efficiency, particularly for gilts destined for the breeding herd. It also suggests that the immunoglobulin immunocrit can be useful in monitoring colostrum ingestion to maximize the beneficial effects of colostrum on subsequent performance.

Effect of fetal size on fetal placental hyaluronan and hyaluronoglucosaminidases throughout gestation in the pig

The trophoblast-endometrial epithelial cell bilayer of porcine placenta undergoes microscopic folding during gestation, and the folded bilayer is embedded in fetal placental stroma. We hypothesized that hyaluronan was a component of fetal placental stroma, and that hyaluronoglucosaminidases played a role in bilayer folding. Gilts were unilaterally hysterectomized-ovariectomized (UHO) at 160 days of age, mated at estrus and killed on days 25, 45, 65, 85 or 105 of gestation. Fetal placental tissues were collected to evaluate hyaluronan and hyaluronoglucosaminidase content. Fetal placental hyaluronan concentration increased (P<0.01) between day 25 and 45 of gestation, remained high throughout gestation, and was greater (P<0.05) in the fetal placenta of the smallest compared to the largest fetuses on day 105 of gestation. Hyaluronan was localized to fetal placental stroma. Three cDNAs for hyaluronoglucosaminidase 1 (two 1379 and one 1552bp) and one cDNA (1421bp) for hyaluronoglucosaminidase 2 were cloned from day-85 fetal placental RNA. Gene expression analysis indicated that the 1379bp form of hyaluronoglucosaminidase 1 mRNA did not differ, the 1552bp form increased, and the 1421bp form of hyaluronoglucosaminidase 2 decreased during pregnancy. Amount of all three mRNAs was greater (P<0.05) in fetal placenta of the smallest compared to the largest fetuses. Zymography indicated 70 and 55kd protein isoforms of hyaluronoglucosaminidase in fetal placental tissue. Both forms increased with advancing gestation and were greater in fetal placenta of the smallest compared to the largest fetuses (P<0.05). These results are consistent with a role for hyaluronan and hyaluronoglucosaminidases in the development of the microscopic folds of the pig placenta during gestation.