Oral administration of domperidone in the first or third week of lactation: Effects on prolactin concentrations and mammary gene expression in sows and piglet growth.

The scope of the present study is endocrine and metabolic control of sow lactation. This project aimed to determine the impact of increasing prolactin concentrations via oral administration of the dopamine receptor antagonist domperidone in the first or third week of lactation in sows. Effects on sow hormonal and metabolic status, lactational performance, and gene expression in mammary epithelial cells were determined. Primiparous sows were divided in 3 treatments: 1) 10 mL of vehicle (table syrup) per os twice daily during the first and third weeks of lactation (Control, CTL, n = 23), 2) 0.5 mg/kg of domperidone per os twice daily during the first week of lactation (LACT1, n = 23), or 3) 0.5 mg/kg of domperidone given per os twice daily during the third week of lactation (LACT3, n = 22). Treated sows also received 10 mL of the vehicle twice daily during the other treatment period. Litter size was standardized to 12 ± 1 and piglets were weighed at birth, 24 h, and on d 8, 15, 22 (weaning), 35, and 56. Sow feed intake was recorded daily. Representative milk samples were obtained on d 7 and 21 of lactation for compositional analyses, and milk fat globules were used to measure mRNA abundances of various genes. Jugular blood samples were obtained from sows on d 1, 7, 14, and 21 of lactation to measure concentrations of prolactin, IGF-1, insulin, urea, and FFA. Concentrations of prolactin were increased (P < 0.01) at the end of the 7-d treatment period with domperidone, whether imposed in the first (LACT1) or third (LACT 3) week of lactation. No other blood variables were affected by treatments and neither was milk composition (P > 0.10). Sow BW, backfat thickness, or feed intake were not altered by treatments (P > 0.10), but piglet BW tended to be greater in litters from LACT3 compared with CTL sows on d 22 and 35 (P ≤ 0.10). Gene expression of EGF in milk fat globules tended to be (LACT1, P < 0.10) or was increased (LACT3, P < 0.05) after treatment, and the effect in LACT1 sows was maintained until d 21 of lactation. The mRNA abundance of SPP1 was increased (P < 0.05) in LACT1 vs CTL sows on d 7, and that of 3 major milk proteins tended to be (CSN1S2 and WAP, P < 0.10) or was greater (LALBA, P < 0.05) in LACT3 vs CTL sows on d 21 of lactation. Oral administration of domperidone during the first or third week of lactation increased prolactin concentrations and altered mRNA abundances of selected genes in milk fat globules. Yet, only the LACT 3 treatment positively affected piglet performance.

Hyperprolactinemia using domperidone in prepubertal gilts: Effects on hormonal status, mammary development and mammary and pituitary gene expression.

Objectives of this experiment were to determine if the domperidone protocol previously used for gestating gilts can also lead to hyperprolactinemia in growing gilts, and to assess the effects of such a protocol on hormonal status, mammary development and gene expression in mammary and pituitary tissue of gilts at puberty. The impact on future lactation performance was also determined. At 75 ± 3 kg body weight (BW), gilts were divided between: 1) controls (CTL), receiving daily intramuscular (IM) injections of canola oil (1.1 mL) for 29 d (n = 41), and 2) treated (DOMP), receiving daily IM injections with 0.5 mg/kg BW of the dopamine receptor antagonist domperidone for 29 d (n = 40). In addition to that daily injection, treated gilts also received twice daily IM injections with 0.5 mg/kg BW of domperidone over the first 3 d of treatment. Fifteen gilts per treatment were sacrificed at 210 ± 5 d of age to collect mammary glands (for compositional analysis and gene expression) and the anterior pituitary (for gene expression). Remaining gilts were bred and allowed to farrow. Blood was sampled at the onset of treatment and on days 14 and 30. Gilts that farrowed were also blood sampled on days 3 and 20 of lactation. Blood was assayed for prolactin (PRL), leptin, insulin-like growth factor 1 (IGF-1), urea, free fatty acids and glucose. Concentrations of PRL increased after 14 d and 30 d of treatment (P < 0.01) and were lesser on day 3 of lactation in DOMP than CTL gilts (P < 0.01). At puberty, there were tendencies (P < 0.10) for total parenchymal protein and DNA to be greater in DOMP than CTL gilts. Treatment did not affect mRNA abundance of PRL or the long form of the PRL receptor genes in the pituitary gland at puberty but expression level of the dopamine receptor D2 and PRL genes was much lower in pubertal than late-pregnant gilts (P < 0.001). Furthermore, many genes related with PRL had a much greater expression level in late pregnancy than at puberty. On day 20 of lactation, CTL sows had greater concentrations of urea than DOMP sows (P < 0.01). The growth rate of litters was not affected by treatment nor was milk composition (P > 0.10). Even though PRL concentrations were increased with treatment, the absence of effect on mammary development was either due to timing relative to developmental stage, whereby treatment was initiated when gilts were too young, or was because all PRL receptors may have been saturated thereby preventing biological action of additional PRL.

Effects of sustained hyperprolactinemia in late gestation on mammary development of gilts.

The objective of this project was to determine the effects of sustained hyperprolactinemia for 7 or 20 d on mammary development in late-pregnant gilts. On day 90 of gestation, gilts were assigned to one of 3 groups to receive intramuscular (IM) injections of (1) canola oil (CTL, n = 18) until day 109 ± 1 of gestation; (2) a dopamine receptor antagonist, domperidone (0.5 mg/kg of body weight [BW]) until day 96 ± 1 of gestation (T7, n = 17); or (3) domperidone (0.5 mg/kg BW) until day 109 ± 1 of gestation (T20, n = 17). Domperidone-treated gilts also received 100 mg of domperidone per os twice daily from days 90 to 93 of gestation. Blood was sampled on days 89, 97, 104, and 110 for prolactin (PRL), insulin-like growth factor 1 (IGF1), lactose, urea, and glucose assays. Mammary glands were collected at necropsy, on day 110, for compositional and cell proliferation analyses. Abundance of mRNA for selected genes was also determined in the mammary gland and the pituitary gland. On day 97 of gestation, PRL concentrations were 3 times greater for T20 and T7 than CTL gilts and were also greater for T20 than T7 and CTL gilts on days 104 and 110 (P < 0.001). Concentrations of IGF1 in T20 and T7 gilts were elevated relative to controls on days 97 and 104 and were greater for T20 vs T7 and CTL gilts on day 110 (P < 0.05). There were no treatment effects (P > 0.1) on parenchymal or extraparenchymal tissue weights, or on epithelial proliferation as measured by immunohistochemistry for Ki-67. Treatments did not alter concentrations of dry matter (DM), fat, or DNA (P > 0.1) in parenchyma. Concentrations of RNA (P < 0.05) and protein (P < 0.10) as well as total parenchymal protein, RNA, and DNA (P < 0.05) were lower, or tended to be, in T20 than T7 or CTL gilts. Hyperprolactinemia for 20 d in late gestation increased mRNA abundance of the milk protein genes beta-casein (CSN2) and whey acidic protein (WAP) (P < 0.05) in mammary parenchyma and also decreased mRNA abundance of the long form of the prolactin receptor (PRLR-LF). Increasing PRL concentrations for 7 or 20 d in late gestation had no beneficial effects on the composition of the mammary gland, and sustained exposure to domperidone for 20 d reduced metabolic activity either by a lower expression of the long form of the PRL receptor in mammary parenchymal tissue or, most likely, by the early involution of parenchymal tissue. In conclusion, results do not support the hypothesis that a sustained hyperprolactinemia in late gestation could enhance mammary development of gilts.

TRIENNIAL LACTATION SYMPOSIUM/BOLFA: Adipokines affect mammary growth and function in farm animals.

The essential role of mammary fat pads in mammary growth and morphogenesis was the first indication that biologically active molecules, secreted from adipocytes or other stromal cells, could regulate endocrine cues for growth and function of the mammary gland. The presence of leptin and adiponectin receptors in mammary tissues suggested that locally produced or circulating adipokines could affect mammary growth and function. Herein, we present the current knowledge on the role of adipokines in mammary cell proliferation and differentiation and in lactogenesis and galactopoiesis in farm animals. We also address the role of milk adipokines in the neonate. Accumulating evidence suggests that adipokines could act as metabolic sensors, regulating mammary growth and function in periods of metabolic adaptations such as late pregnancy and early lactation. Indeed, different experiments reported that adiponectin and leptin expression varies according to physiological stages and nutritional status of the animal. The current review also demonstrates that adipokines, such as leptin and adiponectin, are important regulators of the action of lactogenic hormones in the mammary gland. Findings also suggest important roles for adipokines in growth and intestinal maturation of the neonate.

Differences in body condition of gilts that are maintained from mating to the end of gestation affect mammary development.

The goal of this project was to determine if different body conditions in late gestation that are due to varying body conditions at mating affect mammary development and mammary gene expression of gilts. Gilts that were fed ad libitum in the growing period were selected based on their backfat depths to form 3 groups at mating, namely, low backfat (LBF; 12-15 mm; = 14), medium backfat (MBF; 17-19 mm; = 15), and high backfat (HBF; 22-26 mm; = 16). During gestation, LBF, MBF, and HBF gilts were fed approximately 1.25, 1.43, and 1.63 times maintenance requirements to maintain their differences in body condition. Feed intake was increased by 1 kg in the last 10 d of gestation. Backfat depths of gilts were ultrasonically measured at mating and on d 30, 50, 70, 100, and 109 of gestation. Blood samples were obtained at mating and on d 109 of gestation to measure concentrations of IGF-1, glucose, insulin, estradiol, urea, free fatty acids, leptin, and adiponectin. Gilts were slaughtered on d 110 of gestation to collect mammary glands for compositional analyses. Mammary extraparenchymal tissue weight was lesser in LBF and MBF gilts than in HBF gilts (1,259.3, 1,402.7, and 1,951.5 ± 70.4 g, respectively; < 0.01). The weight of parenchymal tissue was not affected by treatment ( > 0.10), but its composition was altered. Concentrations of DNA and RNA decreased as backfat depth increased ( < 0.05), whereas percent fat and DM increased ( < 0.05). Circulating concentrations of leptin tended to be lower at mating ( < 0.10) and were lower on d 109 of gestation ( < 0.05) in LBF gilts than in HBF gilts. On d 109 of gestation, concentrations of insulin ( < 0.01) and IGF-1 ( < 0.05) were lower in LBF and MBF gilts than in HBF gilts, whereas those of urea were greater ( < 0.05). The mRNA abundance in parenchymal tissue for all genes studied was not affected by treatments ( > 0.10) with the exception of , which had a greater expression level in LBF gilts than in MFB or HBF gilts ( < 0.05). Percent of Ki-67-positive cells, used to assess mammary cell proliferation rate, was greater in HBF gilts than in LBF gilts ( < 0.05). When differences in body conditions of gilts that were present at mating were maintained throughout gestation, it had an impact on mammary development. Extraparenchymal tissue mass was affected and, more importantly, composition of parenchymal tissue was altered, indicating a beneficial effect of gilts being in the thinner treatment groups at mating.

Milk yield, milk composition, and hepatic lipid metabolism in transition dairy cows fed flaxseed or linola.

The response of transition dairy cows to dietary supplementation with fat sources of various fatty acid profiles could affect hepatic fat metabolism differently. Twenty-eight Holstein cows were blocked for similar calving date 4wk before expected parturition to compare the effects of feeding sources of n-3 and n-6 fatty acids on milk production and composition, plasma metabolites, and liver parameters. Cows within each block were assigned to 1 of 3 isonitrogenous and isoenergetic diets: control with a source of calcium salts of palm oil (MEG; 1.1 and 2.6% of the dry matter in prepartum and postpartum diets, respectively); n-3 fatty acids supplied as whole flaxseed (WFL; 4.8 and 7.7% of the dry matter in prepartum and postpartum diets, respectively); and n-6 fatty acids supplied as whole linola (WLO; 4.8 and 7.7% of the dry matter in prepartum and postpartum diets, respectively). Diets were fed until wk 14 of lactation. Contrasts of WFL versus WLO and polyunsaturated fatty acids versus MEG were compared. Cows fed polyunsaturated fatty acids increased dry matter intake over time at a greater extent than those fed MEG, which resulted in enhanced energy balance. Cows fed MEG produced more milk compared with those fed polyunsaturated fatty acids, and there was no difference between those fed WFL and WLO. We found no effect on body condition score and body weight. Plasma concentrations of glucose, fatty acids, and BHB were similar among diets. There was no effect of diet on concentration of glycogen and activities of superoxide dismutase and glutathione peroxidase in the liver. We observed higher concentrations of hepatic lipids and triacylglycerol in cows fed MEG compared with those fed polyunsaturated fatty acids, and no difference between WFL and WLO. Hepatic catalase activity tended to be higher on wk 4 after calving for cows supplemented with WFL compared with those fed WLO. Feeding linoleic and linolenic acids as unprotected oilseeds increased dry matter intake over time at a greater extent for cows fed MEG, improved the energy status, and lowered hepatic lipids and triacylglycerol contents, which may contribute to enhance the health status of transition dairy cows.

Body condition of gilts at the end of gestation affects their mammary development.

The impact of body condition at 110 d of gestation on mammary gland development, mammary gene expression, and hormonal and metabolite status of gilts was studied. Thirty-nine gilts were equally divided into 3 groups based on their backfat thickness at the end of gestation: 1) low backfat (LBF; 12-15 mm), 2) medium backfat (MBF; 17-19 mm), or 3) high backfat (HBF; 21-26 mm). Gilts had similar BW (138.1 ± 8.2 kg) and backfat thicknesses (16.4 ± 1.0 mm) at mating and the 3 groups were achieved via ingestion of varying amounts of feed throughout gestation. Jugular blood samples were obtained from all gilts at mating and at 109 d of gestation to assess hormonal and metabolic statuses, and animals were slaughtered on d 110 to collect mammary glands for compositional analyses and for measure of gene expression. The LBF gilts had less extraparenchymal tissue ( < 0.01) and parenchymal tissue ( < 0.05) than HBF gilts. Mammary parenchyma from LBF gilts also tended to contain less DM ( < 0.10), contained more protein ( < 0.05), and had greater RNA concentrations ( < 0.01) than that from HBF gilts. None of the 15 genes studied in mammary parenchymal tissue differed in terms of expression level, and the rate of mammary cell proliferation was similar among treatments ( > 0.10). There was a tendency for circulating leptin concentrations on d 109 of gestation to be lower in LBF gilts than in MBF gilts ( < 0.10), whereas values for HBF gilts did not differ from those of the other treatments ( > 0.10). Current results demonstrate that being too thin at the end of gestation (12-15 mm backfat) has a negative impact on mammary development in gilts, whereas having backfats varying from 17 to 26 mm seems to have no detrimental effects on mammogenesis. Backfat thickness in late pregnancy must therefore be considered to achieve optimal sow lactation performance.

Delayed muscle development in small pig fetuses around birth cannot be rectified by maternal early feed restriction and subsequent overfeeding during gestation.

Intrauterine variations in nutrient allowance can alter body composition and tissue features of the porcine offspring around birth. This study aimed to determine the effects of fetal weight variations between littermates and of maternal dietary regimen during gestation on fetal muscle traits just before birth. Fourteen pregnant gilts were reared under a conventional (control, CTL; n=7) or an experimental (treatment, TRT; n=7) dietary regimen during gestation. The dietary treatment provided 70% of the protein and digestible energy contents of the CTL diet during the first 70 days of gestation and then, 115% of the protein and digestible energy contents up to farrowing. At 110 days of gestation, sows were sacrificed and one fetus having a low (824±140 g) and one having a normal (1218±192 g) BW per litter were sampled. Irrespective of maternal dietary regimen, the longissimus muscle of the small fetuses exhibited higher expression levels of DLK1/Pref1 and NCAM1/CD56, two genes known to be downregulated during normal skeletal muscle development. Expression levels of the embryonic isoform of the myosin heavy chain (MyHC), both at the mRNA and at the protein levels, were also higher in small fetuses. In addition, the ratios of perinatal to embryonic and of adult fast to developmental MyHC isoforms were generally lower in light fetuses compared with their medium-weight littermates. These modifications suggest a delayed myofiber development in spontaneous growth-retarded fetuses. Finally, GLUT1 was expressed to a lesser extent in the muscle of small v. normal fetuses, suggesting decreased ability for glucose uptake in muscle. Initial feed restriction and subsequent overfeeding of sows during gestation led to a lower expression of the myogenic factor MYOD1, a prerequisite for myogenic initiation in skeletal muscle. This maternal strategy was also associated with a lower expression level of insulin-like growth factor 1 receptor (IGFR) but an upregulation of IGF2. This suggests an altered susceptibility of muscle cells to IGFs' signal in fetuses from treated sows. Altogether, intrauterine growth restriction impaired fetal muscle development, and restricted feeding followed by overfeeding of gestating sows did not allow small fetuses to recover normal contractile and metabolic characteristics.

Effects of the plant extract silymarin on prolactin concentrations, mammary gland development, and oxidative stress in gestating gilts.

The impacts of supplementing the diet of gestating gilts twice daily with 4 g of the plant extract silymarin on circulating hormonal concentrations, oxidative status, mammary development, and mammary gene expression at the end of gestation were determined. Gilts were fed conventional diets during gestation and on d 90 they were assigned as controls (CTL; n = 16) or treated (TRT; n = 17) animals. Treatment consisted of providing 4 g of silymarin twice daily until d 110, at which time all gilts were slaughtered to collect mammary tissue for compositional analyses and measures of gene expression and oxidative status, and liver and corpora lutea for measures of oxidative stress variables. Blood samples for hormonal assays and evaluation of oxidative stress biomarkers were obtained on d 89, 94, and 109 of gestation. Silymarin increased (P = 0.05) circulating concentrations of prolactin over all samples in the repeated in time analysis. In separate analyses for each sampling time, prolactin concentrations in TRT gilts tended (P < 0.10) to be greater than in CTL gilts on d 94 of gestation. Repeated in time analysis also revealed that silymarin reduced (P ≤ 0.05) plasmatic accumulation of biomarkers of oxidative damage to protein (protein carbonyls) between d 89 and 109. There was no effect (P > 0.10) of treatment on progesterone, estradiol, leptin, or 8-hydroxy-2'-deoxyguanosine concentrations. Percent fat in mammary parenchyma was greater (P ≤ 0.05), percent protein was lesser (P ≤ 0.05), and concentrations of both RNA (P ≤ 0.01) and DNA (P < 0.05) were lesser in TRT than CTL gilts. Mammary parenchyma from TRT gilts had lower (P ≤ 0.05) mRNA abundance for STAT5A and leptin and tended to have lower (P ≤ 0.10) abundance for STAT5B than CTL gilts. Silymarin reduced (P ≤ 0.001) protein carbonyls concentrations in liver of TRT gilts. No effect of treatment was observed on antioxidant gene expression and enzymatic activities in liver samples while total superoxide dismutase activity tended to be higher (P ≤ 0.10) in the corpora lutea of TRT animals when compared with CTL. This is the first demonstration that, in female pigs, silymarin can increase prolactin concentrations and protect against oxidative stress, yet the increase in prolactin was not enough to have beneficial effects on mammary gland development in late gestation.

Impact of diet deprivation and subsequent overallowance during gestation on mammary gland development and lactation performance.

The impacts of diet deprivation and subsequent overallowance during gestation on mammary gene expression and development and lactation performance were determined. Gilts were reared under a conventional (control, CTL; n = 59) or an experimental (treatment, TRT; n = 56) dietary regimen during gestation. The experimental regimen provided 70% (restriction diet, RES) and 115% (overallowance diet, OVER) of the protein and DE contents provided by the CTL diet. The RES diet was given during the first 10 wk of gestation followed by the OVER diet until farrowing. Some gilts (14 CTL and 14 TRT) were slaughtered on d 110 of gestation, and the others were allowed to farrow. Of these remaining sows, 28 (14 CTL and 14 TRT) were slaughtered on d 21 of lactation, and the rest underwent a second lactation. At each slaughter, mammary tissue was collected for compositional analyses and assessment of gene expression. Milk samples were collected on d 17 of the first lactation. Litter size was standardized to 11 ± 1, and piglets were weighed weekly until d 18 in both parities. The BW and back fat thickness of TRT first-parity sows were less than those of CTL sows in gestation (P < 0.05), and their BW was also less in lactation (P < 0.05). The BW of TRT second-parity sows was still less at mating (P < 0.05) and tended to be less on d 1 of lactation (P < 0.10) compared with CTL sows. There were no differences in piglet growth between CTL and TRT litters in either parity, yet mammary development and mammary gene expression were affected by treatment. There was less parenchymal tissue (P < 0.01) at the end of the first gestation in TRT than in CTL sows, but parenchymal tissue composition was not altered by treatment. Relative abundance of IGF-1 (P < 0.05), ornithine decarboxylase (P < 0.05), signal transducer and activator of transcription 5B (P < 0.05), and whey acidic protein (WAP, P < 0.01) genes in parenchyma at the end of the first gestation was lower in TRT than in CTL sows, and the effect on WAP genes was still present at the end of the first lactation (P < 0.01). Mammary composition at the end of the first lactation and milk composition were unaffected by treatment. In conclusion, feed deprivation and subsequent overallowance in gestation had unfavorable effects on sow BW, back fat, mammary development, and mammary gene expression at the end of gestation, but piglet growth rate over the 2 parities was not affected.

Effects of leptin and adiponectin on the growth of porcine myoblasts are associated with changes in p44/42 MAPK signaling.

We hypothesized that both adiponectin and leptin affect the growth of porcine skeletal muscle cells, with fatty acids acting as modifiers in adipokine action and that both adipokines influence the gene expression of their receptors. Therefore, the objective of this study was to investigate the effects of recombinant adiponectin and leptin on cell number (DNA) and DNA synthesis rate with and without oleic acid supplementation, on cell death, and on key intracellular signaling molecules of proliferating porcine myoblasts in vitro. Moreover, the mRNA expression of genes encoding for the leptin and adiponectin receptors (LEPR, ADIPOR1, ADIPOR2) as affected by leptin or adiponectin was examined. Recombinant porcine adiponectin (40 µg/mL) and leptin (20 ng/mL) increased DNA synthesis rate, measured as [(3)H]-thymidine incorporation (P < 0.01), reduced cell viability in terms of lactate dehydrogenase release (P < 0.05), or lowered DNA content after 24 h (P < 0.05). In adiponectin-treated cultures, oleic acid supplementation increased DNA synthesis rate and reduced cell number in a dose-dependent manner (P < 0.05). Both adiponectin (P = 0.07) and leptin (P < 0.05) induced a transient activation of p44/42 mitogen-activated protein kinase (MAPK) after 15 min, followed by decreases after 60 and 180 min (P < 0.05). Adiponectin tended to increase c-fos activation (P = 0.08) and decreased p53 activation at 180 min (P = 0.03). Both adiponectin and leptin down-regulated the abundance of ADIPOR2 mRNA and, transiently, of LEPR mRNA (P < 0.05). In conclusion, adiponectin and leptin may adversely affect the growth of porcine myoblasts, which is related to p44/42 MAPK signaling and associated with changes in ligand receptor gene expression.

Milk production in sows from a teat in second parity is influenced by whether it was suckled in first parity.

The impacts of a teat being suckled or not in first parity on its development, gene expression, and milk yield in the next parity were studied. Forty-seven first-parity sows (Sus scrofa) were divided into 2 groups: i) the same teats suckled in 2 subsequent lactations (controls, CTL; n = 22); and ii) different teats suckled in 2 subsequent lactations (treated, TRT; n = 25). In the first lactation, over half of the teats (Teats 1, 2, 5, 6, and 7 from 1 side of the udder, and Teats 3, 4, and 7 from the other side) were sealed with tape so that they were nonfunctional. During the next lactation, the CTL group had the same teats sealed as in the first lactation, whereas the opposite teats were sealed for the TRT group. In both parities, litters were standardized to 7 piglets around birth and to 6 piglets (1 piglet per available teat) at 48 h postpartum. During the second lactation, piglets were weighed at birth and on d 2, 4, 7, 14, 21, 35, and 56 postpartum. Weaning was at 17 ± 1 d of age. Behavioral measures were obtained (using 24-h video recording) on d 3 and d 10 of lactation on 15 sows per treatment to evaluate satiety of piglets, using aggressiveness and nursing behavior as indicators. At weaning in the second lactation, 16 sows per treatment were slaughtered and 4 functional mammary glands were collected for compositional analyses and parenchyma from 2 nonfunctional glands was collected to measure mRNA abundance for selected genes. Piglets from CTL sows weighed 1.12 kg more than piglets from TRT sows (P < 0.05) on d 56, and functional mammary glands from CTL sows contained more parenchymal tissue, more DNA, and more RNA (P < 0.01) than those from TRT sows. The relative mRNA abundance of prolactin in parenchymal tissue tended to be greater in CTL than TRT sows (P < 0.10). Behavioral measures indicated a greater hunger level for piglets using teats that were not previously suckled. Current findings clearly show that teats that were suckled in first lactation produce more milk and have a greater development in the second lactation than nonsuckled teats.

Impact of diet deprivation and subsequent over-allowance during prepuberty. Part 2. Effects on mammary gland development and lactation performance of sows.

The impact of diet deprivation and subsequent over-allowance in prepubertal gilts on their mammary development and mammary gene expression at the end of gestation and their lactation performance over 2 parities was determined. Seventy-seven gilts were reared under a conventional (control, CTL; n = 41) or an experimental (treatment, TRT; n = 36) dietary regimen. The experimental regimen provided 70 (restriction diet, RES) and 115% (over-allowance diet, OVER) of the protein and DE contents provided by the CTL diet. Experimental diets were fed ad libitum starting at 27.7 ± 3.4 kg of BW as follows: 3 wk RES, 3 wk OVER, 4 wk RES, and 4 wk OVER. All gilts were bred, and 34 were slaughtered on d 110 of gestation (18 CTL and 16 TRT) to collect mammary tissue for compositional analyses and gene expression measurements. Remaining gilts (23 CTL and 20 TRT) were maintained for 2 parities, and litter performance data were obtained. Blood samples for hormonal and metabolite assays were obtained on d 110 of gestation from all sows slaughtered at that time and from 14 sows per treatment on d 2 and 17 of lactation in the first parity. Milk samples were obtained from these same sows on d 17 of lactation in both parities. There was a tendency for mammary parenchymal tissue to contain less protein in TRT than CTL sows (P < 0.10), and relative mRNA abundance of the signal transducer and activator of transduction 5B gene was increased in parenchyma from TRT sows (P < 0.05). Circulating prolactin (P < 0.05) and milk lactose concentrations (P < 0.01) were less, whereas milk protein content was greater (P < 0.05) in TRT sows than CTL sows on d 17 of lactation. Nevertheless, growth rate of suckling piglets over the first 2 parities was unaffected by treatment. In conclusion, the use of a diet deprivation and over-allowance regimen in the growing-finishing period did not have beneficial effects on mammary gene expression or on sow and piglet performance.

Impact of diet deprivation and subsequent over-allowance during prepuberty. Part 1. Effects on growth performance, metabolite status, and mammary gland development in gilts.

The impact of diet deprivation and subsequent over-allowance on the metabolite status, mammary development, and mammary gene expression in prepubertal gilts was determined. Forty-seven gilts were reared under a conventional (control, CTL; n = 23) or an experimental (treatment, TRT; n = 24) dietary regimen. The later regimen (consisting of diet deprivation and subsequent over-allowance) provided 70 (restriction diet, RES) and 115% (over-allowance diet, OVER) of the protein and DE contents provided by the CTL diet. Experimental diets were fed ad libitum starting at 27.7 ± 3.4 kg of BW as follows: 3 wk RES, 3 wk OVER, 4 wk RES, and 4 wk OVER. At each diet change, BW and individual feed intakes were measured, and blood samples for metabolite and IGF-I assays were obtained. Some gilts (11 CTL and 12 TRT) were slaughtered on d 235 (after reaching puberty) to collect mammary tissue for compositional analyses and measures of gene expression. Body weight gain (P < 0.01) and G:F (P < 0.05) of gilts were reduced during each period with the RES diet; however, there was no compensatory growth during the periods when the OVER diet was fed. Feeding the RES diet reduced concentrations of urea and IGF-I (P < 0.01) and feeding the OVER diet increased FFA (P < 0.01) and glucose (P < 0.10) in TRT gilts compared with CTL gilts. The TRT gilts had less parenchymal tissue (P < 0.05) and tended to have less total parenchymal fat and protein (P < 0.10) than CTL gilts. The mammary mRNA relative abundance of the signal transducers and activators of transduction 5B was decreased in TRT compared with CTL gilts (P < 0.05). In conclusion, the diet deprivation and over-allowance regimen used in the growing-finishing period did not have beneficial effects on mammary development after puberty. In fact, a detrimental effect was observed.

Effects of supplementary folic acid and vitamin B(12) on hepatic metabolism of dairy cows according to methionine supply.

The present experiment was undertaken to study the interactions between dietary supplements of rumen-protected methionine (RPM) and intramuscular injections of folic acid and vitamin B(12), given from 3 wk before calving to 16 wk of lactation, on hepatic metabolism of lactating dairy cows. Sixty multiparous Holstein cows were assigned to 10 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet calculated to supply Met as 1.83% of metabolizable protein, whereas the 3 other cows were fed the same diet supplemented with 18g of RPM calculated to provide Met as 2.23% of metabolizable protein. Within each level of Met, the cows received no vitamin supplement or weekly intramuscular injections of 160mg of folic acid alone or combined with 10mg of vitamin B(12). Liver biopsies were taken at 2, 4, 8, and 16 wk of lactation. Liver concentrations of folates and vitamin B(12) were increased by their respective supplements but this response to vitamin supplements was altered by methionine supply. Concentrations of total lipids and triglycerides increased in livers of cows fed RPM, whereas concentrations of cholesterol ester, cholesterol, diglycerides, phosphatidylethanolamine, and phosphatidylcholine were not affected. Folic acid, alone or combined with vitamin B(12), tended to increase the ratio of phosphatidylcholine to phosphatidylethanolamine. Gene expression of 5,10-methylene-tetrahydrofolate reductase, microsomal transfer protein, and phosphatidylethanolamine methyltransferase were higher in liver of cows fed RPM supplements. The relative mRNA abundance of 5,10-methylene-tetrahydrofolate reductase and methylmalonyl-CoA mutase were increased by the combined injections of folic acid and vitamin B(12), whereas those of methionine synthase and methionine synthase reductase were not affected by treatments. These results suggest that increasing supply of methyl groups, as preformed labile methyl groups or through methylneogenesis, affected the methylation cycle but had a limited effect on dairy cow performance. The observed effects of the combined supplement of folic acid and vitamin B(12) on lactational performance of dairy cows probably result from an improvement of energy metabolism during early lactation.

Dietary genistein stimulates mammary hyperplasia in gilts.

The possible role of the phytoestrogen genistein on prepubertal development of mammary glands, hormonal status and bone resorption was investigated in gilts. Forty-five gilts were fed a control diet containing soya (CTLS, n = 15), a control diet without soya (CTL0, n = 15) or the CTLS diet supplemented with 2.3 g of genistein daily (GEN, n = 15) from 90 days of age until slaughter (day 183 ± 1). Both basal diets were isonitrogenous and isocaloric. Jugular blood samples were obtained on days 89 and 176 to determine concentrations of isoflavone metabolites (on day 176 only), prolactin, estradiol, progesterone, insulin-like growth factor 1 (IGF1), and N-telopeptide of type I collagen (NTx; on day 176 only). At slaughter, mammary glands were excised, parenchymal and extraparenchymal tissues were dissected, and composition of parenchymal tissue (protein, fat, dry matter (DM), DNA) was determined. Histochemical analyses of mammary parenchyma were performed. Dietary genistein increased parenchymal protein (P < 0.05) while decreasing DM (P < 0.05) and tending to lower fat content compared with the CTLS, but not the CTL0, diet. There was more parenchymal DNA (1.26 v. 0.92 mg/g, P < 0.05) in GEN than CTLS gilts, likely reflecting an increase in the quantity of mammary epithelial cells. Circulating concentrations of genistein were increased in GEN gilts (P < 0.001) but concentrations of hormones or NTx (indicator of bone collagen resorption) were not affected by GEN (P > 0.1). Percentage of estradiol receptor alpha (ERα)-positive epithelial cells was lower (P < 0.05) in GEN than CTLS gilts, whereas 5-bromo-2'-deoxyuridine labeling index was unaltered (P > 0.1). Transcript levels for ERα, ERß, IGF1, epidermal growth factor (EGF), epidermal growth factor receptor and transforming growth factor alpha were not altered by treatments. Supplementation of the diet with genistein during the growing phase in gilts, therefore, led to hyperplasia of mammary parenchymal tissue after puberty; yet, even though circulating genistein was increased, this was not accompanied by changes in mammary expression of selected genes or circulating hormone levels.

Influence of methionine supply on the response of lactational performance of dairy cows to supplementary folic acid and vitamin B12.

The present experiment was undertaken to determine if the effects of supplementary folic acid on lactational performance were caused by improved methylneogenesis and if the supply in vitamin B(12) could affect this metabolic pathway. In this eventuality, supplementary Met, a major source of preformed methyl groups, should reduce the requirements for these vitamins. Sixty multiparous Holstein cows were assigned to 10 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet estimated to supply Met as 1.83% metabolizable protein and 3 cows were fed the same diet supplemented with 18 g of rumen-protected methionine (RPM) to supply Met as 2.23% of metabolizable protein. Within each level of Met, cows received no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid alone or combined with 10 mg of vitamin B(12) from 3 wk before to 16 wk after calving. There was no treatment effect on dry matter intake during pre- and postcalving periods: 13.4 +/- 0.4 and 21.8 +/- 0.4 kg/d, respectively. Milk production was not affected by RPM supplementation. Folic acid and vitamin B(12) given together tended to increase milk production during the 16 wk of lactation. This effect was more pronounced during the first 4 wk of lactation: 37.5, 37.7, and 40.3 +/- 0.9 kg/d for cows receiving no vitamin supplement, folic acid alone, or folic acid combined with vitamin B(12), respectively. Milk fat yield was not affected by treatments. Lactose, crude protein, and total solid yields were greater, in early lactation, in cows injected with folic acid and vitamin B(12) together but this effect diminished as lactation progressed. Intramuscular injections of folic acid alone or combined with vitamin B(12) tended to decrease plasma concentrations of homocysteine from 5.51 microM with no vitamin supplement to 4.54 and 4.77 +/- 0.37 microM, respectively. Results of the present experiment suggest that the effects of the combined supplement of folic acid and vitamin B(12) on lactational performance of dairy cows were not due to an improvement in methyl groups supply, because RPM supplement, a source of preformed methyl groups, did not alter the cow responsiveness to vitamin supplements.

Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows.

The present experiment was undertaken to determine the effects of dietary supplements of rumen-protected methionine and intramuscular injections of folic acid and vitamin B(12), given 3 wk before to 16 wk after calving, on glucose and methionine metabolism of lactating dairy cows. Twenty-four multiparous Holstein cows were assigned to 6 blocks of 4 cows each according to their previous milk production. Within each block, 2 cows were fed a diet estimated to supply methionine as 1.83% metabolizable protein, equivalent to 76% of methionine requirement, whereas the 2 other cows were fed the same diet supplemented daily with 18 g of rumen-protected methionine. Within each diet, the cows were administrated either no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid plus 10 mg of vitamin B(12.) To investigate metabolic changes at 12 wk of lactation, glucose and methionine kinetics were measured by isotope dilution using infusions of 3[U-(13)C]glucose, [(13)C]NaHCO(3) and 3[1-(13)C,(2)H(3)] methionine. Milk and plasma concentrations of folic acid and vitamin B(12) increased with vitamin injections. Supplementary B-vitamins increased milk production from 34.7 to 38.9 +/- 1.0 kg/d and increased milk lactose, protein, and total solids yields. Whole-body glucose flux tended to increase with vitamin supplementation with a similar quantitative magnitude as the milk lactose yield increase. Vitamin supplementation increased methionine utilization for protein synthesis through increased protein turnover when methionine was deficient and through decreased methionine oxidation when rumen-protected methionine was fed. Vitamin supplementation decreased plasma concentrations of homocysteine independently of rumen-protected methionine feeding, although no effect of vitamin supplementation was measured on methionine remethylation, but this could be due to the limitation of the technique used. Therefore, the effects of these B-vitamins on lactation performance were not mainly explained by methionine economy because of a more efficient methylneogenesis but were rather related to increased glucose availability and changes in methionine metabolism.

Characterization of swine adiponectin and adiponectin receptor polymorphisms and their association with reproductive traits.

In this study, polymorphisms in genes encoding porcine adiponectin (ADIPOQ) and its receptors (ADIPOR1 and ADIPOR2) were evaluated for associations with reproductive traits in a Landrace sow population. Sixteen SNPs were identified, and among these, associations were found between reproductive traits and five SNPs. Heterozygous multiparous females for SNP ADIPOQEF601160:c.178G>A had fewer stillborn piglets (P < 0.05) and shorter weaning-to-oestrus intervals (P < 0.05). Multiparous females bearing the mutant allele for SNP ADIPOQEF601160:c.*1094_1095insC gave birth to fewer stillborn piglets (P < 0.05). In addition, selection for the ADIPOQ [A;C] haplotype is expected to result in multiparous sows having the lowest number of stillborn piglets and shorter weaning-to-oestrus intervals. In second-parity sows, the polymorphism in ADIPOR1 (AY856513:c.*129A>C) showed significant associations with live-born (P < 0.01) and stillborn (P < 0.05) piglets. In multiparous sows, a significant association was observed for an ADIPOR2 polymorphism (AY856514:c.*112G>A), with the c.*112GA genotype associated with shorter weaning-to-oestrus intervals (P < 0.01). Haplotype analyses of ADIPOR2 SNPs revealed that selection in favour of the [A;C] haplotype and against the [G;G] haplotype may result in sows having an increased number of live-born piglets and shorter weaning-to-oestrus intervals. We have therefore described specific SNPs and haplotypes that are associated with large litter size, fewer stillborn and mummified piglets and shorter weaning-to-oestrus intervals. Selection for these SNPs and haplotypes is a strategy to improve reproductive success in pigs.

Evaluation of molecular methods used for establishing the interactions and functions of microorganisms in anaerobic bioreactors.

Molecular techniques have unveiled the complexity of the microbial consortium in anaerobic bioreactors and revealed the presence of several uncultivated species. This paper presents a review of the panoply of classical and recent molecular approaches and multivariate analyses that have been, or might be used to establish the interactions and functions of these anaerobic microorganisms. Most of the molecular approaches used so far are based on the analysis of small subunit ribosomal RNA but recent studies also use quantification of functional gene expressions. There are now several studies that have developed quantitative real-time PCR assays to investigate methanogens. With a view to improving the stability and performance of bioreactors, monitoring with molecular methods is also discussed. Advances in metagenomics and proteomics will lead to the development of promising lab-on chip technologies for cost-effective monitoring.