Influence Of Maternal Dietary Enengy Level On Ovary Development Of Fetal And Offspring In Meishan And Yorkshire Gilts

Reproductive productivity in the female pig is determined by proper nutrition level regulating follicle health development which is critical for the ovary to maintain oestrus, fertilization and improving litter size. The primordial follicle of the pig is arising from embryonic phase and regulation of the initial follicle number. In order to understand the time-dependent expression of functional proteins and response to energy levels in fetal ovaries, this study was carried out the differences in fetal ovary development of Meishan and Yorkshire pigs. Furthermore, this study was to test the association of energylevels diet during gestation with ovarian health and development in the offspring.Exp.1 Difference in Yorkshire and Meishan fetal ovarian developmentThe objective of the present study is to test the expression of key proteins that regulate the formation and transformation of primordial follicle in Meishan and Yorkshire porcine breeds. Eight Meishan and eight Yorkshire healthy gilts were used in this experiment. Eight Yorkshire gilts at about 135.54±0.66 kg and eight Meishan gilts with similar weights (72.84±0.66 kg) were mated with identical Yorkshire or Meishan boars. After artificial insemination, the pregnant gilts were housed individually and fed 2 kg/d (0-30 d) or 2.4 kg/d (31-90 d). All diets were formulated to meet nutrient requirements as recommended by the National Research Council (2012) and breeding standards of Chinese pig (2004). Four Meishan and four Yorkshire pregnant gilts were randomly selected on day 55 to be anaesthetized and were slaughtered. The remaining pregnant gilts were similarity slaughtered on day 90 of gestation. The female fetal ovaries of the gilts were immediately excised. The objective of the present study is to test the expression of key proteins offetal ovaries in Meishan and Yorkshire porcine breeds by using two dimensional-difference in gel electrophoresis and MALDI TOF-TOF MS analysis.The results showed that:1. electrophoresis results:1) About 1551 protein spots were detected and 27 individual protein spots were significantly changed (ratioYorkshire/Meishan>1.6 or ratioMeishan/Yorkshire>1.6,P<0.05) on day 55 of gestation in Yorkshire and Meishan fetal ovaries. The proteomic analysis shows that Yorkshire fetal ovaries had 14 up-regulated proteins and 13 down-regulated proteins compared with Meishan fetal ovaries at day 55 of gestation.2) About 1400 protein spots were detected and 16 individual protein spots were significantly changed (ratio Yorkshire/Meishan>1.6 or ratio Meishan/Yorkshire> 1.6, P< 0.05) on day 90 of gestation in Yorkshire and Meishan fetal ovaries. The proteomic analysis shows that Yorkshire fetal ovaries had 7 upregulated proteins and 9 downregulated proteins compared with Meishan fetal ovaries at day 90 of gestation.2. Mass spectrographic analysis results:1) At day 55 of gestation, Yorkshire fetal ovaries express proteins that regulate primordial follicle assembly, such as actin, alpha-1-antitrypsin (AAT), vimentin (VIM), while Meishan fetal ovaries have more proteins play role on uncleaved oocyte stage, such as serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform and, serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform.2) At day 90 of gestation, Yorkshire fetal ovaries express proteins that regulate stress, such as heat shock proteins, while Meishan fetal ovaries have more proteins play role on regulate transformation of primordial follicles, such as alpha-fetoprotein (AFP), and serotransferrin.The results have described several different of proteins participating in the regulation of fetal ovarian development indicating the major mechanisms responsible for the earlier formation of Yorkshire primordial follicles compared with Meishan fetal ovaries and Meishan pigs primordial follicles transformation have a greater developmental potential than the Yorkshire pig.Exp.2 Dietary energy intake affects fetal ovary development in Yorkshire and Meishan pigsThe objective of the present study is to test the dietary energy intake affects fetal ovary development in Yorkshire and Meishan pigs. Sixteen Meishan and sixteen Yorkshire healthy gilts were used in this experiment. Sixteen Yorkshire gilts at about 135.54±0.66 kg and sixteen Meishan gilts with similar weights (72.84±0.66 kg) were mated with identical Yorkshire or Meishan boars. After artificial insemination, Yorkshire pigs were allocated to two energy levels to fed 3.4Mcal/kg or 3.0Mcal/kg and Meishan pigs were allocated to two energy levels to fed 3.0Mcal/kg or 2.6Mcal/kg. Feeding management was similarity to Exp.1. Each group was randomly selected four gilts on day 55 to be anaesthetized and slaughtered. The remaining pregnant gilts were similarity slaughtered on day 90 of gestation. The female fetal ovaries of the gilts were immediately excised. We analyze time-dependent expression of functional proteins in fetal ovaries byproteomic technology, and further study the energy levels of mRNA expression of different proteins determination.The results showed that:1. Proteomic analysis:1) The results showed that there are about 1551 protein spots in Yorkshire fetal ovaries and Yorkshire fetal ovaries on day 55 of gestation had 25 upregulated proteins and 18 downregulated proteins compared with at day 90 of gestation (ratio 55 d/90 d> 1.6 or ratio 90 d/55 d> 1.6, P< 0.05). The expression levels of AFP, AAT, VIM were significantly increased on day 55 of gestation compared with at day 90 of gestation, while gelsolin (GSN),78 kDa glucose-regulated protein (GRP78) were significantly reduced on day 55 of gestation than on day 90 of gestation.2) The results showed that there are about 1740 protein spots in Meishan fetal ovaries and Yorkshire fetal ovaries on day 55 of gestation had 12 up-regulated proteins and 18 down-regulated proteins compared with at day 90 of gestation (ratio 55 d/90 d> 1.6 or ratio 90 d/55 d> 1.6, P< 0.05). The expression levels of AFP, peptidyl-prolyl cis-transisomerase (PIN1) were significantly increased on day 55 of gestation compared with at day 90 of gestation, while AAT, gelsolin were significantly reduced on day 55 of gestation than on day 90 of gestation.2. mRNA expression analysis:1) Those results indicate that the abundance levels of AAT, AFP which regulated primordial follicle formation that were significantly higher (P<0.05) in the low energy levels group on day 55 of gestation than in high energy levels group. The abundance levels of GRP78 which regulated stress resistance ability of the cells, and GSN which regulated primordial follicle transformation to primary follicles that were significantly higher (P<0.05) in the low energy levels group on day 90 of gestation than in high energy levels group.2) The relative mRNA expression levels of AAT and AFP which regulated key proteins of fetal ovary primordial follicle formation that were not altered in different energy levels group (P>0.05) on day 55 of gestation. On day 90 of gestation, in both energy levels PIN1 which regulated inflammatory response and GSN which related to primordial follicle transformation to primary follicles that were expression levels of relative mRNA no altered.The results have described maternal high energy level is not conducive to the fetal ovarian development of Yorkshire pigs by regulated primordial follicle formation, primordial follicle transformation to primary follicles, and tress resistance ability of the cells, but do not affect the fetal ovarian development of Meishan pigs.Exp.3 Effect of energy levels dietary programs during maternal gestation on offspring ovarian healthIn this study we examined the effects of nutritionally induced oxidative stress on follicle development of Yorkshire offspring. Twenty-four pregnant gilts were used in this experiment. Gilts were allocated to two nutrition allowance levels either high-energy (3.4 Mcal/kg) or low-energy diets (3.0 Mcal/kg) level. After artificial insemination, the pregnant gilts were housed individually and fed 2 kg/d (0-30 d),2.4 kg/d (31-90 d) or 3.0 kg/d (91-114 d). Feeding management was similarity to Exp.2. Weaning piglets randomly selected 6of each litter and were given ad libitum access to the same diet. Each group was randomly selected four gilts on day 55 or 90 of gestation, and 160 of offspring, puberty gilts to be anaesthetized and slaughtered. The female fetal ovaries of the gilts were immediately excised anddetermination of relevant indicators. The results showed that:1. the low-energy group had greater large follicle numbers (P<0.05) than the high-energy group on day 160, and the low-energy group showed a marked increased in small follicle numbers on the puberty (P<0.05). Liver weight shows a similar trend in animals on day 160, and was significantly increased (P<0.05) in the low-energy group.2. The MDA concentration was markedly lower (P< 0.05) on day 90 of gestation, but T-SOD, CuZn-SOD, GR, GPx and T-AOC activities were significantly higher (P< 0.05) on day 90 of gestation in the low-energy group rather than in the high-energy group. In addition, T-SOD, GPx and T-AOC activity were significantly higher (P< 0.01) on the low-energy group than in the high-energy group on day 160. The MDA concentration, T-SOD, CuZn-SOD and GPx activity had no significant different at 110kg (P>0.05).3) Furthermore, the apoptosis-related genes of B-cell lymphoma-2 (BCL2) had dramatically higher expression in ovary cells from high-level treated animals on day 90 of gestation and dramatically lower expression of Bcl-2 associated X protein (BAX) (P<0.05) and as shown by real-time PCR analyses. In addition, BAX was significantly increased (P<0.05) in the low-level group than in the high-level group on an offspring day 160, and BCL2 was significantly increased (P<0.05) in the high-level group on day 160 and puberty.The results also revealed thata high level diet may adverse effects on the ovarian development of offspring through regulation of oxidative stressand cell apotosis.In conclusion, our date demonstrated that:the primordial follicle formation is earlier in Yorkshire than in Meishan fetal ovaries, and Meishan pig fetal ovaries have a greater ovaries microenvironment than the Yorkshire fetal ovaries which promoting primordial follicle transformation to primary follicle. The maternal high level diet do no influence the Meishan fetal ovary development. However, a high level diet accelerate cell apoptosis and induce oxidative stress, which have adverse effects on the ovarian health of fetal and offspring. Therefore, the Yorkshire and Meishan gilts have different sensitivities to variations in dietary energy intake levels.