Effect of postpartum collection time and colostrum quality on passive transfer of immunity, performance, and small intestinal development in preweaning calves.

This study evaluated the effects of postpartum collection time and quality of colostrum fed to calves on the failure of passive transfer, growth, and small intestine development in the first 5 wk of life. Newborn calves (Holstein-Friesian × Jersey) were identified at birth and collected either early (E; within 12 h postpartum; n = 20) or late (L; 18-24 h postpartum; n = 20) and fed either high-quality colostrum [HQC, first milking colostrum with Brix% = 23 ± standard deviation (SD) 2] or low-quality colostrum (LQC, mixed colostrum and transition milk with Brix% = 12 ± 1) to create 4 treatments: E-HQC, E-LQC, L-HQC, and L-LQC (n = 10/treatment). After collection, calves (body weight = 32.3 ± 4.6 kg/calf) were fed either HQC or LQC (7.5% of their arrival body weight per feed) for the first 3 (L calves) or 4 feedings (E calves). All calves were then managed and fed similarly using automatic feeders which recorded individual intake of milk replacer and calf starter. Blood samples were taken at d 1 (after collection from dams but before colostrum feeding), 4, 14, and 35 of age to analyze selected metabolites. All calves were killed at d 35 ± 2 of age and histomorphology of duodenum, jejunum, and ileum was evaluated. At collection, 75% of E calves and 58% of L calves had serum total protein ≤52 g/L. At d 4 of age, calves fed HQC had greater serum total protein than calves fed LQC; however, failure of passive transfer (serum total protein ≤52 g/L) incidence did not differ between HQC and LQC. Collection time did not affect the scouring duration, but the amount of electrolyte used to treat sick calves was lower in L versus E calves, whereas feeding HQC versus LQC lowered both the scouring duration and electrolyte use to treat sick calves. Calves fed HQC had a greater total surface area of the duodenum (+23%) and jejunum (+17%) compared with LQC calves. Duodenal crypts were deeper in E-LQC calves than E-HQC and L-HQC calves, whereas L-LQC calves were intermediate. Villus height to crypt depth ratio in duodenum, jejunum, and ileum was greater in HQC than LQC calves. A trend toward greater average daily gain was observed in HQC versus LQC calves (667 vs. 590 g/d) but the average daily gain was not influenced by collection time. Serum IGF-1 at d 4 was higher in HQC versus LQC calves and this might have contributed to greater average daily gain and small intestine development. Calves fed HQC had higher feed conversion ratios (FCR; total body weight gain/total dry matter intake) compared with LQC calves, and L calves had higher FCR compared with E calves. In conclusion, in comparison to feeding LQC, feeding HQC reduced the scouring duration, enhanced surface area of duodenum and jejunum, and improved FCR during the first 5 wk of calf age. Postpartum collection time of calves did not affect small intestine development, but L calves had higher FCR and required a lesser volume of electrolytes to treat scours compared with E calves during the first 35 d of life.

Effect of arginine or glutamine supplementation and milk feeding allowance on small intestine development in calves.

The development of the small intestine (SI) is important for the health and growth of neonatal calves. This study evaluated the effect of arginine (Arg) and glutamine (Gln) supplementation and 2 levels of milk allowance on the histomorphological development of the SI in preweaning calves. Sixty mixed-sex Friesian × Jersey calves (3-5 d of age) were offered reconstituted whole milk (125 g/L, 26% fat, 26% protein) at either high (20% of arrival body weight/d; HM) or low (10% of arrival body weight/d; LM) milk allowance without (Ctrl) or with supplementary Arg or Gln (at 1% of milk dry matter) in a 2 × 3 factorial design (n = 10/treatment). After 35 d on the diets, all calves were slaughtered to collect tissues for examination of SI development. Calves in the HM group had higher milk intake, total weight gain, and average daily gain compared with LM calves, but no effect of AA supplementation nor an interaction between milk allowance and AA supplementation was observed. For the duodenum, we observed an AA by milk allowance interaction for villus height and width, and goblet cell number per villus (HM-Arg > HM-Gln > HM-Ctrl), and villus height to crypt depth ratio (HM-Arg > HM-Gln = HM-Ctrl), but no effect of AA supplementation in the LM group. Goblet cell numbers per 100 µm of SI were greater in Arg-supplemented calves than in unsupplemented controls, with Gln-supplemented calves intermediate to but not different from the other groups. Epithelium thickness was greater in LM than in HM calves. Villus density, crypt depth, and muscle thickness did not differ between groups. For the jejunum, there was an AA by milk allowance interaction for villus height, villus surface area, and villus height to crypt depth ratio (HM-Arg = HM-Gln > HM-Ctrl), with no effect of AA supplementation in the LM groups. Amino acid supplementation affected goblet cell number per villus (HM-Gln > HM-Ctrl calves, HM-Arg intermediate), and both LM-Arg and LM-Gln calves had greater numbers than LM-Ctrl calves. Villus width, crypt depth, and muscle thickness were greater in HM than LM calves but there was no effect of AA supplementation. Villus density, goblet cell number per 100 µm of SI, and epithelium thickness were unaffected by AA supplementation and milk allowance. Milk allowance and AA supplementation had no effect on SI morphology in the ileum. Increasing milk allowance improved villus height, width, and surface area but only in Arg- or Gln-supplemented calves, not in control calves. The observed changes in development may be important for intestinal functionality, integrity, and barrier function in preweaning calves, potentially through increased cell growth and proliferation or reduced levels of cellular atrophy.

Invited review: impact of specific nutrient interventions during mid-to-late gestation on physiological traits important for survival of multiple-born lambs.

To improve production efficiency, the sheep meat industry has increased flock prolificacy. However, multiple-born lambs have lower birth weights, increased mortality and reduced growth rate compared with single-born lambs. Lamb mortality is a major issue for livestock farming globally and solutions are required to increase survival to realise the value of increased flock fecundity. Nutrition during gestation can influence maternal-foetal placental nutrient transfer and thus foetal growth and organ/tissue development, as well as improve postnatal productivity. This review covers the challenges and opportunities associated with increased prolificacy, highlights gaps in our knowledge and identifies some opportunities for how targeted intervention with specific nutrients during mid-to-late pregnancy may influence lamb survival and productivity with a specific focus on pasture-based systems. This time frame was selected as intervention strategies in short-time windows post-pregnancy scanning and before lambing to improve lamb survival in high-risk groups (e.g. triplets) are likely to be the most practical and economically feasible options for pasture-based extensive farming systems.

Intravenous maternal -arginine administration to twin-bearing ewes, during late pregnancy, is associated with increased fetal muscle mTOR abundance and postnatal growth in twin female lambs.

The aims of this study were to determine whether parenteral Arg administered to well-fed twin-bearing ewes from 100 to 140 d of pregnancy influences fetal skeletal muscle growth, the abundance and activation of mechanistic target of rapamycin (mTOR) protein, and postnatal muscle growth of the offspring. Ewes fed 100% of NRC-recommended nutrient requirements for twin-bearing ewes were administered an intravenous bolus of either 345 µmol Arg HCl/kg BW or saline solution (Control) 3 times per day. At 140 d of pregnancy (P140), a group of 11 Control and 9 Arg-treated ewes were euthanized and hind leg muscles and longissimus dorsi (LD) were excised and weighed. A sample of LD was snap frozen in liquid nitrogen for later analysis of free AA (FAA) concentration, mTOR abundance and phosphorylation, and biochemical indices (DNA, RNA, and protein content). For the remaining 25 ewes (Arg, = 13, and Control, = 12), Arg administration was continued until the initiation of parturition and ewes were allowed to lamb. Lambs were weaned at postnatal Day 82 and grazed on pasture until postnatal day 153 (PN153), when a subset of 20 lambs ( = 10 per group) was euthanized. At P140, only the psoas major was heavier in the Arg-administered group compared with the Control group. Female lambs from ewes supplemented with Arg (Arg-F) had increased abundance of total mTOR, RNA concentration, and RNA:DNA ratio in LD compared with female lambs from Control ewes (Con-F), whereas males did not differ. At PN153, Arg-F were heavier than Con-F and had heavier LD and plantaris and a trend for heavier psoas major muscles compared with Con-F. In contrast, BW and individual muscle weights did not differ in male lambs. Lambs from Arg-treated ewes had heavier semimembranosus and tended to have heavier biceps femoris compared with Control lambs. The RNA concentration in LD was greater in Arg-F compared with Con-F, and DNA concentration was greater in the Arg group compared with the Control group. In conclusion, Arg administration to the ewe during gestation increases female lamb weight and muscle weight after birth and these changes are associated with altered mTOR protein abundance and have potential implications for sheep production.

Mammary transcriptome analysis of lactating dairy cows following administration of bovine growth hormone.

The galactopoietic effect of growth hormone (GH) in lactating ruminants is well established; however the mechanisms that mediate these effects are not well understood. The first objective of this study was to determine the effect of GH on the synthesis of the major casein and whey proteins. The second objective was to identify the genes and pathways that may be involved in mediating the effect of GH on milk synthesis. A single subcutaneous injection of a commercially available slow release formulation of GH (Lactatropin®), or physiological saline solution (control) was administered to non-pregnant dairy cows (n=4/group) in mid-late lactation. Milk samples were collected for composition analysis and mammary lobulo-alveolar tissue was collected postmortem 6 days post injection. Gene expression profiles were evaluated using either a 22 000 bovine complementary DNA microarray or quantitative PCR (qPCR), and microarrays were validated by qPCR. The yield of all the major casein and whey proteins was increased 32% to 41% in GH-treated cows, with the exception of α-lactalbumin yield which was elevated by 70% relative to controls. Treatment with GH treatment tended to increase the concentration of α-lactalbumin but had no effect on the concentration of any of the major milk proteins. Messenger RNA (mRNA) abundance of the major whey and casein genes, with the exception of α-s2-casein, was increased in response to GH compared with controls, which is consistent with the positive effect of GH on milk production. Treatment with GH treatment influenced the mRNA abundance of genes involved in cell growth and proliferation, transcriptional and translational regulation, actin cytoskeleton signalling, lipid metabolism and cell death. This study has provided new insights into the cell signalling that may be involved in mediating the effect of GH on milk production in the mammary gland of lactating dairy cows.

Intravenous maternal -arginine administration to twin-bearing ewes during late pregnancy enhances placental growth and development.

This study aimed to investigate if intravenous maternal Arg administration to well-fed twin-bearing ewes, from 100 to 140 d of gestation or birth, could enhance placental development and placental nutrient transport. Ewes received intravenous infusions of saline (control) or 345 µmol Arg HCl/kg of BW 3 times daily from d 100 of pregnancy (P100) to d 140 of pregnancy (P140; cohort 1) or from P100 to birth (cohort 2). At P140, ewes in cohort 1 were euthanized and individual placentae per fetus were dissected and placentomes were classed per type (A to D) and size (light to heavy). Placentome number and individual weight were recorded. As an indicator of placental nutrient transport, blood plasma was collected from the uterine ovarian vein (UOV), uterine artery (UA), and umbilical vein and artery at the time of euthanasia and analyzed for metabolites and free AA concentrations. The ewes in cohort 2 were allowed to lamb and lambs were weighed at birth. The expelled placenta was dissected and number of cotyledons and weights of total cotyledons, remaining fetal membranes, and total placenta were recorded. At P140, Arg-infused ewes had a 63% ( = 0.03) greater number of unoccupied caruncles than control ewes. No differences were observed for placental weight at P140. At birth, lambs from Arg-infused ewes tended to have 11% ( = 0.09) greater placental weight and 34% ( = 0.03) greater total cotyledon weight compared with control lambs. Arginine-infused ewes (Arg-infused) had increased concentrations of Arg ( = 0.0001) and ornithine (Orn; = 0.004) but decreased concentrations of Met ( = 0.01) and His ( = 0.02 and = 0.09, respectively) compared with control ewes in plasma UOV and UA. Fetuses from Arg-infused ewes had increased concentrations of Orn ( = 0.005) and decreased concentrations of His ( = 0.006), Met ( = 0.003), and Lys ( = 0.01) but no differences in Arg ( > 0.10) concentrations were found compared with control fetuses in umbilical artery and vein plasma. This study showed that maternal Arg administration of well-fed twin-bearing ewes during late pregnancy tended to improve placental growth and development.

Identification of amino acids associated with skeletal muscle growth in late gestation and at weaning in lambs of well-nourished sheep.

The objective of this study was to determine the association between intracellular free AA (FAA) profiles in skeletal muscle with muscle growth in twin and singleton fetuses in late pregnancy and at weaning, under an ad libitum feeding regime of the dam. Plasma from singleton- (n = 9) and twin-bearing (n = 10) ewes at d 140 of pregnancy and FAA in the semitendinosus muscle (STM) from the corresponding fetuses were studied. At weaning, intracellular STM FAA concentrations were compared between twins at the same age as singletons (Twin(age); n = 17) and at the same weight as singletons (Twin(wt); n = 17) to that of singletons (n = 20). Twin fetuses were 15% lighter (P = 0.03) with a 20% lighter STM (P = 0.02) compared to singletons. Maternal plasma FAA were similar (P ≥ 0.17) between singleton- and twin-bearing ewes. Twin fetuses had greater (P < 0.05) plasma concentrations of glutamine, histidine, and methionine and lower (P < 0.05) concentrations of aspartate, citrulline, glutamate, and ornithine compared with singletons. In fetal STM, twins had lower (P < 0.05) concentrations of aspartate and valine and greater (P < 0.01) concentration of methionine. Correlations were found between fetal STM weight and intracellular concentrations of arginine (r = 0.66, P < 0.01) and glutamine (r = 0.49, P < 0.01). Compared to singletons at weaning, Twin(age) were 16% lighter (P < 0.01) and the STM weight was proportionately 16% lighter (P < 0.01). For Twin(wt), the magnitude of the difference for STM weight was reduced to 8% lighter (P = 0.02). Compared to singletons, Twin(age) lambs had greater (P < 0.05) intracellular concentrations of glutamine, histidine, threonine, asparagine, alanine, serine, and glutamate but reduced taurine. The differences in FAA concentrations were less between Twin(wt) and singletons than between Twin(age) and singletons. Positive correlations were found between leucine, lysine, methionine, phenylalanine, proline, threonine, and tyrosine muscle concentration and STM weight at weaning. Males differed from females in intracellular FAA both in late pregnancy and at weaning. Twins had reduced RNA content during pregnancy and at weaning, suggesting a lower capacity for protein accretion. These data suggest that specific FAA concentrations are associated with differences in muscle growth during late pregnancy, notably arginine and glutamine, and reduced protein synthesis capacity. However, the relevance of specific FAA varies according to stage of development and sex of the lamb.

Increased milk protein synthesis in response to exogenous growth hormone is associated with changes in mechanistic (mammalian) target of rapamycin (mTOR)C1-dependent and independent cell signaling.

The objective of this study was to determine if increased milk protein synthesis observed in lactating dairy cows treated with growth hormone (GH) was associated with mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) regulation of downstream factors controlling nucleocytoplasmic export and translation of mRNA. To address this objective, biochemical indices of mammary growth and secretory activity and the abundance and phosphorylation status of mTORC1 pathway factors were measured in mammary tissues harvested from nonpregnant lactating dairy cows 6 d after treatment with a slow-release formulation of GH or saline (n=4/group). Treatment with GH increased mammary parenchymal weight and total protein content and tended to increase ribosome number and cell size, whereas protein synthetic efficiency, capacity, and cell number were unchanged. Cellular abundance of the mTORC1 components mTOR and (phosphorylated) mTOR(Ser2448) increased, as did complex eukaryotic initiation factor 4E:eukaryotic initiation factor 4E binding protein 1 (eIF4E:4EBP1), whereas no change was observed for mTORC1-downstream targets 4EBP1, 4EBP1(Ser65), p70/p85(S6K) and p70(S6K)Thre389/p85(S6K)Thre412. Changes in activation were not observed for any of the targets measured. These results indicate that GH treatment influences signaling to mTORC1 but not downstream targets involved in the nucleocytoplasmic export and translation of mRNA. Increased eIF4E:4EBP1 complex formation indicates involvement of the mitogen-activated protein kinase (MAPK) pathway. Abundance of MAPK pathway components eIF4E, eIF4E(Ser209), eIF4E:eIF4G complex, MAP kinase-interacting serine/threonine-protein kinase 1 (MKNK1), MKNK1(Thr197202), and ribosomal protein S6 kinase, 90kDa, polypeptide 1 (RPS6KA1) increased significantly in response to GH, whereas relative activation of the proteins was unchanged. Expression of IGFBP3 and IGFBP5 increased, that of IGF1R decreased, and that of IGF1 remained unchanged in response to GH. PatSearch analysis of the milk caseins αS1-casein, αS2-casein, and ß-casein, MAPK signaling target RPS6KA1, and proliferation gene IGFBP3 mRNA indicated that all contained putative eIF4E-sensitivity elements. In response to GH, these genes were all upregulated, suggesting that increased abundance of eIF4E and eIF4E(Ser209) plays a role in mediating their nucleocytoplasmic export. We propose that, in response to GH, the IGF1-IGF1R-MAPK signaling cascade regulates eIF4E-mediated nucleocytoplasmic export and translation of mRNA, whereas mTOR controls cell renewal, cell turnover, and rRNA transcription through an alternative signaling cascade.

Placental nutrient transport is affected by pregnancy rank in sheep.

Understanding the link between placental function and fetal growth is critical to comprehend the mechanisms underlying altered fetal growth. This study investigated the relationship between fetal weight and placentome type and size in placentae of singleton and twin fetuses and fetuses within a twin pair from ad libitum-fed ewes at d 140 of pregnancy. In addition, insulin, IGF-I, metabolites, and free AA profiles in fetal, umbilical artery, and vein plasma of singleton and twin fetuses were investigated and used as an indicator of placental nutrient transport. Individual placentae per fetus were dissected, placentomes were classed per type (A to D) and size (light to heavy), and placentome number and individual weight were recorded. Twin fetuses were 16% lighter (P = 0.01) than singletons and had a smaller placenta, with 28% decreased placentome weight (P = 0.03) and 35% fewer placentomes (P = 0.001). Twins also had a different distribution of placentome type and size compared with placentae of singletons, such that twins showed a greater proportion of type B and light placentomes compared with singletons. In twins, umbilical artery plasma had less Glu (P < 0.05) and greater Gln (P < 0.05) concentrations than fetal plasma or umbilical vein plasma, but no differences in AA concentrations were observed between these pools in singletons. Glutamate is a major oxidation energy source for the placenta, and the fetal liver is the net producer of Glu using Gln as its main precursor, indicating that the functionality of the fetoplacental unit may be different between singletons and twins. Twin fetuses had 13% less insulin (P = 0.04) concentrations in umbilical artery plasma than singletons. plasma of twin fetuses had 39% less IGF-I (P = 0.003), 33% less His (P = 0.03), and 22% less Gln (P = 0.02) concentrations and tended to have 44% less Arg (P = 0.07) and 20% less Leu (P = 0.06) concentrations than singletons. Arginine, His, and Leu are examples of AA that can promote insulin secretion, and in turn, insulin can increase fetal IGF-I concentrations. In addition, insulin and IGF-I are important fetal growth factors by stimulating and regulating AA transport across the placenta. Collectively, these results indicate that the functionality of the fetoplacental unit may be different between singletons and twins and that AA transport may be reduced in twin placentae.

Genetic variation in sperm production.

In boars, the primary determinant of daily sperm production is the number of Sertoli cells, which establishes testicular weight. The only breed comparison of foetal testicular development in boars contrasted two diverse breeds, White composite (WC, Landrace-Yorkshire) with Meishan, a Chinese breed that undergoes pubertal development at a young age and has small testicular size. During the prenatal period, the pattern of change in testicular development is similar in these two breeds with both having their greatest proportion of proliferating Sertoli cells at 90 days of gestation, and with WC boars possessing more Sertoli cells and greater mass of seminiferous tubules during the latter half of gestation. During the first month of life, Meishan boars accumulate Sertoli cells and mass of seminiferous tubules at a greater rate than WC boars, and Meishan boars undergo terminal differentiation of Sertoli cells at a younger age. Postpubertal boars, within each breed and crossbreds of the two breeds, with small testicular size have increased circulating concentrations of follicle-stimulating hormone. No direct breed comparisons of testicular development are apparent for postpubertal boars of other breeds. Accepting the limitations of data reported from different laboratories, Piau boars reach puberty at an older age and have a greater proportion of their testes occupied with seminiferous tubules than Meishan boars; both breeds have small testes. A gene or genes on the X chromosome code for small testicular size in Meishan crossbred boars; genetic determinants of testicular size and sperm production in other breeds remain to be identified.

Stereological evaluation of Sertoli cell ontogeny during fetal and neonatal life in two diverse breeds of swine.

Chinese Meishan (MS) boars have smaller testes due to fewer Sertoli cells compared with White Composite (WC) boars. The objective was to describe Sertoli cell development relative to circulating FSH concentrations in fetal and neonatal MS and WC boars. Testes and blood samples were collected on days 60, 75, 90 and 105 postcoitum (dpc) and 1, 7, 14 and 25 postpartum (dpp). One testis was immunostained for GATA4 or Ki67 antigen to evaluate total and proliferating Sertoli cell numbers respectively. Testicular size was greater (P<0.01) in WC than MS boars at all ages, associated with a greater mass of interstitial tIssue. Tubular mass (P<0.01) was greater in prenatal WC boars, but postnatally increased more rapidly (P<0.001) in MS boars, exceeding WC boars by 25 dpp. Sertoli cell numbers increased with age, was greater (P<0.001) in WC than MS boars during prenatal development but increased rapidly (P<0.01) by 1 dpp in MS and thereafter was similar in both breeds. The proportion of Ki67-positive Sertoli cells was maximal at 90 dpc, declining thereafter, did not differ between breeds through 7 dpp, but was greater (P<0.05) in WC than MS boars at 14 and 25 dpp. Plasma FSH concentrations were greater (P<0.05) in WC than MS boars at 75 dpc. FSH concentrations were elevated at 105 dpc (MS) and 1 dpp (WC) but declined thereafter with advancing postnatal age in both breeds. This study illustrates that late gestation represents the period of maximal Sertoli cell proliferation. Despite asynchronous Sertoli cell population growth between breeds during early postnatal life, differential mature Sertoli cell numbers and testicular size are probably due to differences in duration of the proliferative period after 25 dpp, potentially regulated by Sertoli cell maturation and blood-testis barrier formation. These events were not associated with fetal or early postnatal changes in FSH secretion.

Endocrine and molecular influences on testicular development in Meishan and White Composite boars.

The aim of this study was to evaluate developmental changes in thyroid hormone and other key endocrine hormones/molecular markers produced by testicular cells, in relation to breed differences in proliferation and maturation of Sertoli cells and general testicular morphological development in Meishan (MS) and White Composite (WC) boars. Blood samples and testes were collected on days 60, 75, 90 and 105 post coitum (dpc) and days 1, 7, 14 and 25 post partum (dpp). Testes were immunostained for thyroid hormone receptor-beta1 (THRbeta1), GATA4, Müllerian-inhibiting substance (MIS), 17-alpha-hydroxylase (P450(c17)) and inhibin subunits (alpha, betaA, betaB). In addition, protein levels were determined by densitometry. Plasma concentrations of free triiodothyronine (T(3)) were greater in MS (hyperthyroid) compared with WC (hypothyroid) boars (P<0.01) during fetal life, but the reverse was evident postnatally. Elevated levels of free T(3) during fetal life were associated with increased levels of THRbeta1, suggesting increased thyroid responsiveness of the testis during this time, contrasting with observations during early postnatal life. Localization patterns of THRbeta1, MIS, GATA4 and the inhibin subunits were consistent with previous studies. MIS protein levels declined more rapidly (P<0.001) in MS compared with WC Sertoli cells postnatally, consistent with earlier maturation of Sertoli cells as indicated by our previous study. In this study, transient neonatal hyperthyroidism in MS boars during late gestation was associated with a decline in proliferation and early maturation of Sertoli cells, followed by early onset of puberty in this breed. These observations indicate a possible role for thyroid hormone in the modification of Sertoli cell development, thereby influencing growth and differentiation of the testis in pigs.

Expression levels of Mullerian-inhibiting substance, GATA4 and 17alpha-hydroxylase/17,20-lyase cytochrome P450 during embryonic gonadal development in two diverse breeds of swine.

Sexual differentiation and early embryonic/fetal gonad development is a tightly regulated process controlled by numerous endocrine and molecular signals. These signals ensure appropriate structural organization and subsequent development of gonads and accessory organs. Substantial differences exist in adult reproductive characteristics in Meishan (MS) and White Composite (WC) pig breeds. This study compared the timing of embryonic sexual differentiation in MS and WC pigs. Embryos/fetuses were evaluated on 26, 28, 30, 35, 40 and 50 days postcoitum (dpc). Gonadal differentiation was based on morphological criteria and on localization of GATA4, Mullerian-inhibiting substance (MIS) and 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450(c17)). The timing of testicular cord formation and functional differentiation of Sertoli and Leydig cells were similar between breeds. Levels of GATA4, MIS and P450(c17) proteins increased with advancing gestation, with greater levels of MIS and P450(c17) in testes of MS compared with WC embryos. Organization of ovarian medullary cords and formation of egg nests was evident at similar ages in both breeds; however, a greater number of MS compared with WC embryos exhibited signs of ovarian differentiation at 30 dpc. In summary, despite breed differences in MIS and P450(c17) levels in the testis, which may be related to Sertoli and Leydig cell function, the timing of testicular differentiation did not differ between breeds and is unlikely to impact reproductive performance in adult boars. In contrast, female MS embryos exhibited advanced ovarian differentiation compared with WC embryos which may be related to the earlier reproductive maturity observed in this breed.

An integrated comparative map of the porcine X chromosome.

The objectives of this study were to assign both microsatellite and gene-based markers on porcine chromosome X to two radiation hybrid (RH) panels and to develop a more extensive integrated map of SSC-X. Thirty-five microsatellite and 20 gene-based markers were assigned to T43RH, and 16 previously unreported microsatellite and 15 gene-based markers were added to IMpRH map. Of these, 30 microsatellite and 12 gene-based markers were common to both RH maps. Twenty-two gene-based markers were submitted to BLASTN analysis for identification of orthologues of genes on HSA-X. Single nucleotide polymorphisms (SNPs) were detected for 12 gene-based markers, and nine of these were placed on the genetic map. A total of 92 known loci are present on at least one porcine chromosome X map. Thirty-seven loci are present on all three maps; 31 loci are found on only one map. Location of 33 gene-based markers on the comprehensive map translates into an integrated comparative map that supports conservation of gene order between SSC-X and HSA-X. This integrated map will be valuable for selection of candidate genes for porcine quantitative trait loci (QTLs) that map to SSC-X.

Temporal and spatial localization patterns of Gata4 during porcine gonadogenesis.

The zinc finger transcription factor Gata4, is associated with gonadal development in many species. The present study characterizes temporal and spatial localization of Gata4 throughout gonadogenesis in porcine embryos. Immunohistochemical studies illustrated that Gata4 protein is present in the coelomic epithelium prior to histological differentiation of the nascent bipotential gonad, marking the future site of both XX and XY porcine gonads. Many somatic cells of both XX and XY bipotential gonads continue to retain Gata4 immunoreactivity throughout sexual differentiation and subsequent gonadal development. Testicular cords were evident by 26 days postcoitum. Gata4 was present in Sertoli cells, identified by virtue of coexpression with Müllerian inhibiting substance and also interstitial cells including Leydig cells throughout fetal and postnatal life. Many somatic cells of the differentiating ovary including follicular cells also contained Gata4 protein throughout fetal and postnatal life. Gata4 was not present in germ cells, endothelial cells, or other undifferentiated mesenchymal cells of both XX and XY gonads. A population of Gata4-positive cells in the dorsal mesentery was continuous with the coelomic epithelium of the gonad. This localization pattern led to the hypothesis that a subpopulation of somatic cells in the dorsal mesentery moves toward the gonad. An in vitro cell migration assay demonstrated that Gata4-positive cells preferentially migrate toward explanted gonadal tissue, and morphological features of the developing gonad supported this hypothesis. This study illustrates that Gata4 is a very early marker for gonad formation, highlights species differences in temporal and spatial localization patterns, and suggests a potential role for Gata4 in the development of both XX and XY porcine gonads. Further, we suggest that mesenchymal cells of the dorsal mesentery may provide a source of somatic cells that migrate and incorporate into the gonad and contribute to various somatic cell lineages. Overall, the spatial and temporal localization patterns of Gata4 during porcine gonadogenesis implies a much earlier and wider role for Gata4 than previously reported in other species.

Immunohistochemical detection of myogenic cells in muscles of fetal and neonatal lambs.

Previous studies have implied that myonuclei accumulation in a muscle is more important than myofibre number in the determination of muscle size in fetal/neonatal lambs. However, due to the lack of a reliable marker, the role of myogenic precursor nuclei (satellite cells) in myofibre hypertrophy in late fetal and postnatal life is not well understood. In this study, MyoD was shown to be a useful marker for actively proliferating satellite cells in both fetal and neonatal lambs. MyoD was used to determine whether there were differences in the number of actively proliferating satellite cells between single and twin fetuses/neonates, which may explain at least some of the difference in myofibre size observed near birth. Eighteen single-bearing and 9 twin-bearing Coopworth ewes were randomly assigned to one of three slaughter groups (100, 120 and 140 days of gestation). The remaining ewes were kept on pasture until 20 days postpartum at which time 4 single and 4 twin lambs were sacrificed. Twin fetuses/neonates had lower body weights and muscle weights compared to singles. Lower muscle weights in the twins were associated with smaller myofibre cross-sectional areas and lower total nuclei numbers and myogenic precursor cell numbers per muscle in selected hind-limb muscles. These results indicate that myofibre hypertrophy in late gestation and early postnatal life is related to myogenic precursor cell number which may have important implications for growth potential of the growth-restricted fetus.

Specific staining of Sertoli cell nuclei and evaluation of Sertoli cell number and proliferative activity in Meishan and White Composite boars during the neonatal period.

The positive relationship between Sertoli cell number and testicular size emphasizes the importance of determining factors involved in the regulation of the Sertoli cell population. Based on data from other species and indirect evidence in the boar, it is generally accepted that porcine Sertoli cells proliferate rapidly throughout the early postnatal period. However, direct evaluation of Sertoli cell number and the proliferative activity of Sertoli cells during the early postnatal period in boars have not been reported. Stereological enumeration of Sertoli cells is a labor-intensive process and would be greatly facilitated by a marker for these cells especially in the sexually mature male. Thus, the first objective of this study was to determine if expression of the transcription factor GATA-4 is an effective marker for fetal, postnatal, and adult Sertoli cells to facilitate enumeration procedures. The second objective was to evaluate the proliferative activity and growth of the Sertoli cell population in neonatal White Composite and Meishan boars, known to differ in mature testis size and Sertoli cell number, to determine the importance of this developmental period for the adult Sertoli cell population. GATA-4 was abundantly expressed by Sertoli cells throughout fetal and prepubertal stages of development and specifically stained both type A and B Sertoli cell nuclei in the sexually mature boar. Immunoreactivity was never observed in the germ cells regardless of their stage of development, illustrating that GATA-4 is a useful marker for both developing and adult Sertoli cells in the boar. Testicular size did not differ between breeds on Day 1 postpartum, but by 14 days postpartum White Composite boars had significantly larger testes compared to Meishan boars (P: < 0.001). Similarly, Sertoli cell number did not differ between breeds at 1 day postpartum; however, at 14 days postpartum White Composite boars had a significantly larger Sertoli cell population compared to Meishan boars (P: < 0.05). Surprisingly, despite having more Sertoli cells than Meishan boars at 14 days postpartum, the proportion of actively proliferating Sertoli cells in the White Composite boars was almost 50% less than the Meishan boars. This result illustrates that rapid rates of Sertoli cell proliferation probably occurred prior to 14 days postpartum in the White Composite boars. Collectively, these results illustrate that the relationship between testicular size and Sertoli cell number is manifested very early in the postnatal period for these two breeds. The substantial difference in the size of the Sertoli cell population and their proliferative activity between Meishan and White Composite boars during the early postnatal period emphasizes the importance of this early period for the establishment of the Sertoli cell population and subsequent adult testicular size.

Muscle growth, cell number, type and morphometry in single and twin fetal lambs during mid to late gestation.

Muscle growth, myofibre number, type and morphometry were studied in large hindlimb muscles of single and twin fetal lambs during mid to late gestation. Placental insufficiency, evident by lower total placentome weight and number per fetus, resulted in reduced fetal weights from 100 to 140 days gestation in twins compared with singletons (at 140 days: 5016 +/- 108 g v. 5750 +/- 246 g, respectively; P<0.05). However, competition between littermates did not consistently reduce muscle mass (15-22%) until 140 days gestation. Apparent myofibre number increased with age, indicating that the full complement of myofibres in some large hindlimb muscles may be achieved during early postnatal life. Litter size did not impact on apparent myofibre number in the semitendinosus, plantaris or gastrocnemius muscles. However, a transient effect on myofibre number in the adductor femoris muscle was observed from 80-120 days gestation. The phenotypic maturation of myofibres was unaffected by increasing litter size. Smaller muscle mass in twins was associated with smaller myofibre cross-sectional area in the semitendinosus, adductor femoris and gastrocnemius muscles at 140 days gestation. A similar trend was observed for the plantaris muscle. These results indicate that while competition between littermates for nutrients in late gestation can impact on both fetal and muscle mass, the fetus has the capacity to buffer against the effects of restricted nutrient supply on myofibre hyperplasia and phenotypic maturation, but myofibre hypertrophy is compromised.

Morphometric analysis of myofibre development in the adductor femoris muscle of single and twin fetal lambs.

Cellular development of the adductor femoris muscle from twin and single fetuses was studied at 140 days gestation to evaluate the effect of moderate fetal growth retardation on myofibre development. Twin fetuses had lower bodyweights (13%) and disproportionately small adductor femoris muscle weights (22%) compared with single fetuses. Reduced muscle mass was associated with smaller myofibre cross-sectional areas (CSA) and lower DNA content (22%), indicative of fewer myonuclei and retarded myofibre hypertrophy. Myofibre number and the phenotypic maturation of the myofibres were similar between twins and singletons. These results indicate that even modest growth restriction during fetal life can negatively influence myofibre hypertrophy, highlighting the importance of fetal nutrition for muscle growth. Large muscles, such as the adductor femoris, have intrafascicularly terminating myofibres, which necessitates accurate sampling of the muscle when investigating possible perturbations in morphological characteristics (e.g. between singletons and twins). The second objective of the present study was to investigate the impact of the sampling site on the morphological parameters of the adductor femoris muscle. The apparent total myofibre number decreased from the proximal to the distal region of the adductor femoris muscle. The apparent number of slow-twitch fibres also decreased from the proximal to the medial region, but was not different between the medial and distal regions of the muscle. Similarly, myofibre CSA differed between the medial and distal regions. These results indicate that, particularly with large muscles, such as the adductor femoris, which has intrafascicularly terminating myofibres, single site sampling for the determination of morphological fibre characteristics may generate misleading results and that careful selection of the sampling area may be necessary.

Maternal constraint influences muscle fibre development in fetal lambs.

The objective was to examine myogenesis in two situations expected to be characterized by maternal constraint: (i) in fetuses due to be born in spring (n=10) or autumn (n=10); and (ii) in single (n=16) and twin (n=20) fetal lambs. Maternal constraint operating through limitation of placental size, as measured by placentome weight per fetus, was evident in each study. Although a lower placental weight did not influence body and muscle weights of fetuses due to be born in the spring or autumn, twins had lower body and muscle weights than singles. Fibre number and average fibre cross-sectional (CS) area were differentially affected by season and fetal number. The differences in muscle fibre morphology between spring- and autumn-born fetuses suggest that muscle fibre development was influenced by maternal constraint in the absence of an effect on fetal weight. The differences in muscle fibre number and CS area in particular muscles from twin and single fetuses suggest that more severe maternal constraint, reflected in a lower placental size per fetus, not only influences fetal weight but can also affect muscle development.