Maternal energy insufficiency affects testicular development of the offspring in a swine model.

We determined the effects of insufficient maternal energy on testicular development in offspring in a swine model. Thirty-six sows were divided into control (CON) and low-energy diet (LE) groups during gestation. We observed that the number of Sertoli, germ, and Leydig cells in the offspring of the CON group were significantly higher than those in the LE group at 28 and 120 d after birth. Furthermore, the percentage of apoptotic testis cells was significantly higher in the offspring of the LE group than in the CON group. Transcriptome analysis of differentially expressed mRNAs and long noncoding RNAs in offspring testes indicated that these RNAs were mainly involved in lipid metabolism, apoptosis, cell proliferation, and some pivotal regulatory pathways. Results revealed that AMPK-PI3K-mTOR, MAPK, and oxidative phosphorylation signaling pathways play an important role in mediating the programming effect of insufficient maternal energy on testicular development, and that this effect occurs mainly at an early stage in life. mRNA and protein expression analyses confirmed the importance of certain signaling pathways in the regulation of testicular development. This study provides insights into the influence and possible mechanism underlying the effect of inadequate maternal energy intake on testicular development in the offspring.

Comparison of microRNA transcriptomes reveals differential regulation of microRNAs in different-aged boars.

To elucidate the role of microRNA (miRNA) during early testicular development, we constructed three small RNA libraries from boar testes at three timepoints. Utilizing Solexa deep sequencing technology, over 12 million reads were measured. Of a total of 263 known miRNAs, 159 were co-expressed in all libraries. Read counts of the top 20 most abundant miRNAs accounted for more than 75% of total known miRNAs, and in all libraries, miR-10b was the most abundant. Analysis of higher fold changes in miRNAs (|log2_fold changes| > 4) revealed seven miRNAs that were involved in testes development (miR-381, miR-205, miR-217, miR-146a-5p, miR-187, miR-215, and miR-195). Among these, miR-146a-5p and miR-195 played an important role in 0-day-old (N0) and 28-day-old (N28) boars; miR-187 and miR-205 primarily regulated reproductive processes in N28 and 120-day-old (N120) boars; and miR-381, miR-217, and miR-215 participated in the modulation of testes development during all three timepoints. These miRNAs regulated testes cell proliferation and apoptosis at different stages. Furthermore, the predicted miRNA targets from the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that these differentially expressed miRNAs likely regulated developmental processes via PI3K-AKT, mitogen-activated protein kinase, and other related signaling pathways. Our results present a genome-wide miRNA profile from the testes of different-aged boars and provide a useful theoretical basis for further studies on the role of miRNA regulation in testes development during early life.

Effect of intra-uterine growth restriction on long-term fertility in boars.

The present study assessed the effect of birthweight on reproductive performance, including a possible mechanism, in male pigs. Ten newborn male piglets, including five normal birthweight (NBW) piglets and five intra-uterine growth restricted (IUGR) piglets, were used in the study. All piglets were weaned on Day 28 and fed the same diet during the experiment (10 months). Average daily weight gain, feed intake and the feed conversion ratio were higher in NBW than IUGR piglets. Similarly, testis volume and the number of Leydig and Sertoli cells in the distal portion of the testes were higher in NBW than IUGR piglets (P<0.05). Semen volume (P<0.05) and the total number of spermatozoa per ejaculate (P=0.08) were lower in IUGR boars. Testosterone concentrations on Day 141 and prostaglandin E2 concentrations on Days 82 and 141 were higher in IUGR than NBW boars (P<0.05). The malondialdehyde content of seminal plasma was higher in IUGR boars, whereas sperm glutathione peroxidase activity was lower in IUGR versus NBW boars (P<0.05). Expression of DNA methyltransferase (Dnmt) genes Dnmt1, Dnmt3a, histone-lysine N-methyltransferase (Suv39h2), and lysine (K)-specific demethylase Kdm4a was upregulated in testes from IUGR boars. These findings suggest that growth restriction affects sperm production via reproductive organ development and epigenetic regulation.

Effect of dietary supplementation with amino acids on boar sperm quality and fertility.

The aim of this study was to evaluate the effects of dietary supplementation with amino acids on sperm quality and fertility rates after insemination with boar semen. Twelve Yorkshire boars were paired by age and allocated to one of two dietary treatments composed of total lysine levels of 0.64% (T1) and 0.96% (T2), with the lysine: methionine: threonine: tryptophan: valine ratio in the diets set to 100:27:73:19:69 through the addition of synthetic amino acids. Semen was collected twice weekly (phase 1, 1-12 wk); every other day (phase 2, 13-16 wk); twice weekly (phase 3, 17-26 wk); and daily (phase 4, 27-28 wk). Semen was collected from boars during phase 3 and used to inseminate 64 multiparous sows. Our results showed that sperm concentration and total sperm cells were greater in boars in T2 than in boars in T1 in phases 2 and 4 (P<0.05). Sperm motility parameters, morphologically normal sperm, and acrosome integrity in T2 boars were greater than those in T1 boars (P<0.05) during the experiment. Free amino acid concentrations in seminal plasma increased in T2 boars (P<0.05). Furthermore, sows inseminated with semen collected from T2 boars gave birth to more live piglets than those inseminated with semen collected from T1 boars (P=0.04). In conclusion, supplementation of boar diet with amino acids improves sperm quality, and subsequently increases fertilization capacity and the number of live piglets.

Effects of different dietary n-6/n-3 polyunsaturated fatty acid ratios on boar reproduction.

BACKGROUND: N-3 and N-6 polyunsaturated fatty acids are widely used in reproduction, yet few studies have addressed the effects of dietary n-6/n-3 ratios on boar reproduction. The present study aimed to determine the effects of different dietary n-6/n-3 ratios on the reproductive performance of breeding boars. Thirty-two boars with body weights of 15.0 ± 1.4 kg were divided into four treatments (C, T1, T2, T3) and fed diets with different n-6/n-3 fatty acid ratios (29.06:1, 20.07:1, 1:1, 1:17.96, respectively) for 174 days. RESULTS: The highest testis index was observed for treatment T2. Sperm density and total sperm number per ejaculate in the T2 treatment were significantly higher than those in all other treatments, whereas the sperm deformity rate was the lowest. Interestingly, the fatty acid compositions and ratios of sperm were consistent with dietary treatments. Acid phosphatase and fructose concentration of seminal plasma, and the total superoxide dismutase and glutathione peroxidase of sperm in T2 were higher than those in other treatments. The concentration of testosterone and prostaglandin E2 increased in boars fed on diets supplemented with fatty acids as compared with boars subjected to the C group treatment, reaching a peak at n-6/n-3 fatty acid ratios of 1:1. Furthermore, higher expression of Δ(6)-fatty acid desaturase and peroxisome proliferator activated receptor-α in spermatozoa of the T2 treatment were observed, indicating more vigorous metabolism and intensive hormonal regulation. CONCLUSIONS: Our data suggest that the ideal n-6/n-3 ratio in the diet of breeding boars is 1:1, and proper balancing of n-6/n-3 fatty acids plays an important role in male reproduction.

Effect of different amino acid patterns on semen quality of boars fed with low-protein diets.

The objective of the present study was to determine the effect of different amino acid patterns on the semen quality of boars fed with low-protein diets. Twenty-four boars were randomly divided into 4 groups (HP, LP1, LP2, and LP3). HP boars received 17% crude protein diet with a lysine:threonine:tryptophan:arginine (Lys:Thr:Trp:Arg) ratio of 100:50:20:104. Other boars received 13% CP and similar Lys levels (0.84%) with Lys:Thr:Trp:Arg ratios of 100:50:20:71, 100:76:38:71, and 100:76:38:120 for LP1, LP2, and LP3, respectively. These results showed sperm motility in the LP3 group was higher than in HP group during the 13-22 week period. The total sperm number, acrosome integrity ratio, and the effective total sperm number in LP3 and LP2 was higher than in other groups, and the abnormality ratio was lower than in other groups during the 13-18 week period. During 19-22 week period, in LP1 and LP3 groups, total sperm number and effective total sperm number were higher than in other groups, abnormality ratio was lower, and acrosome integrity ratio was higher than in the HP group. Nitric oxide synthase activity of seminal plasma and nitric oxide concentration of spermatozoa were significantly higher in the LP3 group than in other groups. Furthermore, mRNA expression of androgen receptor in testes was up-regulated in LP3. In conclusion, we suggest that the optimum ratio of Lys:Thr:Trp:Arg in a 13% CP diet for boars is 100:76:38:120, which results in similar or better reproductive performances than a 17% CP diet.

Use of sodium butyrate as an alternative to dietary fiber: effects on the embryonic development and anti-oxidative capacity of rats.

In this study, we evaluated the effect of replacing dietary fiber with sodium butyrate on reproductive performance and antioxidant defense in a high fat diet during pregnancy by using a rat model. Eighty virgin female Sprague Dawley rats were fed one of four diets--(1) control diet (C group), (2) high fat + high fiber diet (HF group), (3) high-fat +5% sodium butyrate diet (SB group), and (4) HF diet + α-cyano-4-hydroxy cinnamic acid (CHC group)--intraperitoneally on days 8, 10, 12, 14, and 16 of gestation. SB and dietary fiber had similar effects on improving fetal number and reducing the abortion rate; however, the anti-oxidant capacity of maternal serum, placenta, and fetus was superior in the HF group than in the SB group. In comparison, CHC injection decreased reproductive performance and antioxidant defense. Both dietary fiber (DF) and SB supplementation had a major but different effect on the expression of anti-oxidant related genes and nutrient transporters genes. In summary, our data indicate that SB and DF showed similar effect on reproductive performance, but SB cannot completely replace the DF towards with respect to redox regulation in high-fat diet; and SB might influence offspring metabolism and health differently to DF.

Effect of different dietary omega-3/omega-6 fatty acid ratios on reproduction in male rats.

BACKGROUND: Beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs) on the reproduction of male animals are widely described in the literature. However, there is little information about the effect of n-3/n-6 PUFA ratios on male health and reproduction. The aim of this study was to investigate the effects of diets with different n-3/n-6 PUFA ratios on the reproductive performance of male rats. METHODS: Eighty male Sprague Dawley (SD) rats were supplemented with diets containing different n-3/n-6 PUFA ratios (0.13, 0.40, 0.85, 1.52 and 2.85) for 60 days. Half of the rats in each group were sacrificed on day 60, and the other half were chosen to mate with female mice to assess the effects of n-3/n-6 ratios on reproductive performance. RESULTS: Sperm density and sperm motility of the 1.52 group were higher than other groups (P < .05), and the development of testis and the morphological structure of sperm in the 1.52 group were better than other groups. Furthermore, a higher litter size and birth weights of offspring were observed in the 1.52 group. Additionally, serum reproductive hormone levels were significantly affected by the n-3/n-6 ratios. CONCLUSION: These findings demonstrated that a balanced n-3/n-6 ratio was important in male rat reproduction. Therefore there is a necessity to determine an appropriate n-3/n-6 PUFA ratio in man and different male animals in the future.

Effect of maternal dietary energy types on placenta nutrient transporter gene expressions and intrauterine fetal growth in rats.

OBJECTIVE: The objectives of this study were to investigate the effects of maternal dietary energy types on the mRNA expressions of the placental nutrient transporter and intrauterine fetal growth and to examine whether altered intrauterine fetal growth could be associated with different gene expressions relating to fetal energy metabolism and DNA methylation. METHODS: Seventy-two 3-mo-old rats were allocated to one of four groups: low fat/low fiber (L-L), low fat/high fiber, high fat/low fiber (H-L), or high fat/high fiber. Rats were fed the treatment diets 4 wk before mating and continued in pregnancy until sample collections were obtained on days 13.5 and 17.5 of pregnancy. RESULTS: The fetal weight in the L-L group was significantly lower than that in the H-L group (P < 0.05). The placental nutrient transporter mRNA expressions of glucose transporter-3 (Slc2a3) and cationic amino acid transporter-1 (Slc7a1) in the L-L group with a decreased fetal weight were downregulated compared with that in the H-L group with an increased fetal weight. However, placental Slc2a1 and the system A amino acid transporter gene Slc38a4 mRNA expressions were adaptively upregulated by the L-L diet with a decreased fetal weight (P < 0.05). For the placental imprinted gene Igf-2 and H19 expressions, lower Igf-2 and higher H19 expressions were associated with the decreased fetal growth in the L-L group compared with the H-L group with an increased fetal weight. Different fetal growth was associated with different DNA methyltransferase-1 and methyltransferase-3a expressions (P > 0.05) and energy metabolism-related genes. CONCLUSION: Collectively, these results demonstrated that intrauterine fetal growth could be affected by different energy intake types through placenta nutrient transporter gene expressions, and different fetal growths were associated with altered fetal genes related to DNA methylation and energy metabolism.

The beneficial effect of fiber supplementation in high- or low-fat diets on fetal development and antioxidant defense capacity in the rat.

BACKGROUND: There is mounting evidence that an imbalance in oxidant/antioxidant activities plays a pivotal role in fetal development. AIM OF THE STUDY: To determine the effects of maternal intake of fat and fiber on fetal intrauterine development and antioxidant defense systems of rats. METHODS: Virgin female Sprague-Dawley rats were randomly assigned to 4 groups according to diet: the low-fat, low-fiber group (LL); the low-fat, high-fiber group (LH); the high-fat, low-fiber group (HL); and the high-fat, high-fiber group (HH). The diets were fed 4 weeks prior to breeding through day 17.5 of pregnancy. Dietary intakes of fiber (wheat bran and oat) and fat were quantitatively varied, while intakes of energy and essential nutrients were kept constant among the diets. RESULTS: Rats fed a fiber-rich diet had significantly improved fetal numbers, as well as enhanced activity of superoxide dismutase (SOD) and capacity of scavenging free radicals (p < 0.05). Meanwhile, the placental malondialdehyde and protein carbonyl levels were affected by the diet fat and fiber levels (p < 0.05). Compared with the LL group, the mRNA abundance of hypoxia-inducible factor 1α (HIF-1α) and thioredoxin-2 (Trx2) in the maternal liver and glutathione peroxidase 1 (GPx1) in the placenta and fetus were significantly downregulated in the HL group (p < 0.05). Furthermore, rats fed a fiber-rich diet had significantly upregulated mRNA expressions of Cu,Zn-SOD, Mn-SOD, and HIF-1α in the maternal liver (p < 0.05); Cu,Zn-SOD and Mn-SOD in the placenta (p < 0.05); and Cu,Zn-SOD in the fetus (p < 0.05). CONCLUSION: When energy intakes are equivalent, consumption of fiber in high- or low-fat diets benefits fetal development and growth, through improvements in maternal, placental, and fetal antioxidant defense capacities.

Beneficial effects of dietary fibre supplementation of a high-fat diet on fetal development in rats.

The objective of the present study was to investigate the effects of the addition of fibre and the antioxidant N-acetylcysteine (NAC) to fat-rich diets on fetal intrauterine development in rats. A total of eighty virgin female Sprague-Dawley rats were fed a control diet, a high-fat diet (HF), a high-fat and high-fibre diet (HFF) or a high-fat NAC diet until day 19·5 of gestation. Maternal HFF consumption resulted in a significantly higher mean fetal number and placental weight than in the other groups (P < 0·05). The HFF diet significantly abrogated HF-induced decreases in maternal serum and placental superoxide anion and hydroxyl radical scavenging capacities (P < 0·05); partially abrogated HF-induced increases in maternal serum and placental malondialdehyde (MDA) and protein carbonyl concentrations (maternal serum MDA and placental protein carbonyl, P < 0·05); resulted in significantly higher fetal liver total superoxide dismutase (SOD), Cu- and Zn-containing SOD and Mn-containing SOD (Mn-SOD) activities than in the HF group (P < 0·05). Furthermore, mRNA expressions of hypoxia-inducible factor 1-α, thioredoxin 2 and Mn-SOD in fetal liver and Mn-SOD in fetal heart and placental GLUT3 in the HFF group were higher than those in the other groups (P < 0·05). The inclusion of dietary fibre in the HF diet was more effective than NAC supplementation in maintaining maternal serum and placental superoxide anion and hydroxyl radical scavenging capacities close to those of the control. These results suggest that maternal fibre intake during pregnancy is beneficial for fetal intrauterine development possibly through the improvement of maternal, placental and fetal antioxidant capacities and placental nutrient transfer capacity.

Vitamin a supplements alleviate inflammatory responses in reproductive tracts of male mice infected with pseudorabies virus.

Vitamin A is largely thought to have immune potential for mammal health; however, no conclusive mechanisms exist regarding its role in the pathogen-initiated innate immune response, or in the linkage between the innate and adaptive immune system during sperm formation in the male reproductive tract. Therefore, this study was conducted to evaluate the nutritional protective effect of vitamin A supplementation on reproductive performance and immune function of the male mouse challenged with pseudorabies virus (PRV). Sperm quality, testis toll-like receptors (TLRs) mRNA expression levels, and serum concentration of cytokines and immunoglobulins at 7 or 14 days post-injection were compared between control mice and PRV-challenged mice fed the same diet supplemented with vitamin A at 0, 4000, 10,000, 25,000 and 50,000 IU/kg, respectively. PRV- and phosphate buffered saline (PBS)-injection were performed when the mice in the unsupplemented group were marginally deficient in vitamin A. Sperm quality (sperm density and deformity ratio) of PRV-injected mice was significantly harmed by PRV, but this effect was attenuated by increased vitamin A consumption. Vitamin A supplements also attenuated PRV-challenge-induced increase in testis TLR3, TLR7, and TLR9 mRNA expression and serum pro-inflammatory cytokine (gamma interferon, IFN-gamma; and interleukin 1-beta,IL-1beta) concentration, and decrease in serum anti-inflammatory cytokine (IL-10) concentration. Higher than normal vitamin A consumption was recommended to counteract the deleterious effects of viral invasion, possibly through the downregulated expression of TLRs, and thus to improve immunity and reproductivity of male mice challenged with an invading pathogen.

Effects of vitamin E on reproductive protection in pregnant mice infected with pseudorabies virus (PRV) via regulating expression of Toll-like receptors (TLRs) and cytokine balance.

Vitamin E supplement and pseudorabies virus (PRV) infection have a reciprocal role in influencing the maternal immune response, a key determinant of the success or failure of pregnancy. However, it remains unknown whether vitamin E supplement provides protection against PRV-induced failure of pregnancy. This study was therefore conducted to investigate the effect of dietary vitamin E level (0, 75, 375, 750 and 1,500 mg/kg) on the reproduction performance, immunity and expression of Toll-like receptors (TLRs) of PRV-challenged mice. The mortality and abortion rate of PRV-challenged mice decreased with the increase in vitamin E consumption. Overall, PBS-injected mice had a higher live embryo number and live litter size than PRV-challenged mice. Both live embryo number and live litter size of PRV-challenged mice increased with increasing vitamin E levels. Vitamin E supplement resulted in decreased concentration of serum IL-2 and IFN-γ, but increased concentration of serum IL-10. The concentration of serum IgG, IgA and IgM increased with increasing vitamin E levels. In the uterine and embryo mRNA abundance of TLR3, TLR7 and TLR9 was higher in PRV-challenged mice than that in PBS-injected mice fed on the same dosage of vitamin E. The mRNA abundance of embryonic TLR3, TLR7 and TLR9 in PRV-challenged mice decreased with increasing vitamin E levels. Collectively, vitamin E supplement may improve reproductive performance of PRV-challenged mice by attenuating PRV-induced negative effects on the cytokine profile, immunoglobulin synthesis and TLR expression.