Effects of single nucleotide polymorphisms in the 5'-flanking region of heat shock protein 70.2 gene on semen quality in boars

This study aims to elucidate the effects of single nucleotide polymorphisms (SNPs) in the 5'-flanking region of porcine heat shock protein 70.2 gene (HSP70.2) on semen quality in boars. Genomic DNA isolated from 55 boars (41 Duroc, nine Landrace, and five Yorkshire) was subjected to PCR amplification of the 5'-flanking region of HSP70.2. The nucleotide sequences were determined by automated sequencing. Five SNPs (sites 44, 232, 250, 345, and 393) were detected in this region. Semen quality was evaluated in terms of sperm motility, percentage of normal sperm, percentage of sperm with proximal plasma droplet, percentage of abnormal sperm, sperm concentration, semen volume per ejaculate and total sperm number per ejaculate. The effect of the SNPs on semen quality was evaluated based on breed-corrected data within a season. During the cool season, the sperm motility of boars with AA genotype at the 232 site was significantly higher than that of boars with CC genotype (P<0.05). Meanwhile, boars with AC genotype at the 232 site had higher total sperm number per ejaculate than did those with CC genotype. In the hot season, heterozygotes at both the 232 and 250 sites had significantly higher total sperm number of per ejaculate than AA homozygotes (P<0.05). Semen volume of boars with TT and TC genotypes at the 345 site was significantly larger than that of those with CC genotype (P<0.05). Meanwhile, semen quality for boars with TT genotype at the 345 site was significantly higher than that of boars with TC or CC genotype (P<0.05), that is the semen contained higher percentages of normal sperm and lower percentages of abnormal sperm or sperm with proximal plasma droplets. Results herein suggest that the SNPs in the 5'-flanking region of porcine HSP70.2 are associated with semen quality traits in the hot season.     

母猪哺乳初期的母性行为与催乳素受体基因多态性关系的初探

应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)方法对民猪和长白猪的催乳素受体(Prolactin receptor, 简称PRLR)基因进行了单核苷酸多态性分析, 以研究PRLR基因影响母猪母性行为性状的可能性。结果发现在PRLR基因的cDNA第1620位点处存在NaeⅠ多态性。测序表明, 该点存在一个T→C的单碱基突变, 属沉默突变。对3种基因型与部分行为性状进行最小二乘分析, 结果表明, AB型母猪与其他两种基因型母猪相比, 表现出较低的侧卧转为其他姿势的频率以及由母猪结束哺乳的频率(P<0.05), 其他行为性状在基因型间不显著。因此, 推测等位基因A可能是影响母猪母性行为的不良基因。     

PRLR、RBP4基因与苏姜猪繁殖性能的相关性分析

采用PCR-RFLP方法对71头苏姜猪的PRLR基因、RBP4基因的多态性进行了检测,分析不同多态性间繁殖性能的差异,探讨将PRLR基因、RBP4基因作为标记辅助选择用于苏姜猪核心群选育的可行性,从DNA水平上为苏姜猪的选育提供技术性支持.结果表明:PRLR、RBP4两个墓因位点在苏姜五世代母猪中均存在多态性.在该试验中,PRLR基因、RBP4基因各自基因型的效应差异均不显著(P>0.05),合并基因型的效应显著(P<0.05),可以联合标记用于苏姜猪核心群的选育.     

Candidate gene markers for sperm quality and fertility of boar

Candidate genes gonadotropin releasing hormone receptor (GNRHR), prolactin (PRL), prolactin receptor (PRLR), follicle-stimulating hormone beta (FSHB), luteinizing hormone beta (LHB), follistatin (FST), inhibin alpha (INHA), inhibin beta A (INHBA) and inhibin beta B (INHBB) were investigated for their association with sperm quality traits of sperm concentration (SCON), motility (MOT), semen volume per ejaculate (VOL), plasma droplets rate (PDR), abnormal sperm rate (ASR) and fertility traits of non return rate (NRR) and number of piglets born alive (NBA). The experimental material included 356 boars of Pietrain (PI) and Pietrain x Hampshire (PI x HA). Analysis of variance revealed significant association of GNRHR with MOT (P = 0.0161), PDR (P = 0.0048) and ASR (P = 0.0201), INHBA was found to have significant effects on PDR (P = 0.0318) and ASR (P = 0.0067), INHBB was significant (P = 0.0360) for SCON trait. FSHB, FST, INHA, PRL, PRLR and LHB had no significant effects on any trait in this experiment.     

Effects of <Emphasis Type="Italic">Mbo</Emphasis>II and <Emphasis Type="Italic">BspM</Emphasis>I polymorphisms in the gonadotropin releasing hormone receptor (<Emphasis Type="Italic">GnRHR</Emphasis>) gene on sperm quality in Holstein bulls

The hypothalamic gonadotropin-releasing hormone receptor (GnRHR) plays an essential physiological role in reproductive function, which triggers the synthesis and release of luteinizing hormone and follicle stimulating hormone in the pituitary. The objective of this study was to investigate the effects of polymorphisms of GnRHR gene on the quality of fresh and frozen semen in Holstein bulls. The PCR-RFLP method was applied to detect G286A and T340C transitions determining MboII and BspMI polymorphisms, respectively, in the exon I of bovine GnRHR gene and evaluated its associations with sperm quality traits in 131 Holstein bulls. In polymorphic locus 286, bulls with the GA genotype had significantly higher sperm motility in frozen semen (FMOT) than GG genotype (P < 0.01). In polymorphic locus 340, bulls with heterozygote CT genotype had significantly higher sperm motility (MOT), semen volume per ejaculate (VOL), and lower abnormal spermatozoa rate (ASR) than homozygote TT genotype (P < 0.05). Bulls contained one A allele or C allele had a favorable, positive effect on sperm quality traits. These results indicate that GnRHR gene can be a potential marker for improving sperm quality traits, and imply that bulls with GA or CT genotype should be selected in breeding program.     

Growth hormone gene polymorphism and reproductive performance of AI bulls

Relationships between the growth hormone gene RFLP polymorphism and bull sperm characteristics were the objects of the present study. DNA was extracted from blood or sperm samples collected from 113 AI bulls and submitted for polymerase chain reaction (PCR) followed by digestion with Alu I restriction enzyme. The bGH genotypes were visualized on 10% polyacrylamide gel. The analyzed population of AI bulls consisted of dairy (Holstein Fresian [HF] crossbred [HF x Polish Black and White]) and beef breeds (Limousine, Charolaise, Piemontese, Angus and Hereford). The frequency of the Leu allele was 0.86 among dairy bulls and 0.38 in beef bulls (0.14 and 0.62 for the Val allele, respectively). Eight sperm characteristics and Day 60 non-return rates (NRR) were analyzed. The 3 genotype groups (LL, VV and LV) and the effect of production type (dairy or beef) on sperm characteristics were considered. None of the traits showed significant variability in relation to the bGH genotype, although a tendency was observed for LL bulls to have a lower ejaculate volume and VV bulls higher NRR. Moreover some statistically significant associations with production type were noticed: beef bulls were superior in sperm concentration and non-return rate, whereas dairy bulls excelled in individual fresh sperm motility.     

Prolactin Receptor as a Candidate Gene for Prolificacy of Small Tail Han Sheep

DNA polymorphism of the ovine prolactin receptor gene (PRLR) was investigated and used to study its effect on litter size in sheep. By means of PRLR gene sequence homology between sheep and human, three primer pairs were designed for polymerase chain reaction (PCR) amplification within intron 1 and exon 10 of the PRLR gene in sheep. In these parts of the gene the single nucleotide polymorphisms were detected by PCR-single strand conformation polymorphism (SSCP) in 314 Small Tail Han ewes. These poly-morphisms were used to study the associations with litter size. The results indicated that there were three genotypes (AA, AB and BB) detected by three primer pairs. For three primer pairs the frequency of allele A was 0.96, 0.79, 0.68; and the frequency of allele B was 0.04, 0.21, 0.32, respectively. The frequency of genotype AA was 0.93, 0.62, 0.51; the frequency of genotype AB was 0.06, 0.34, 0.34; the frequency of genotype BB was 0.01, 0.04, 0.15, respectively. The Small Tail Han ewes with genotype BB or AB had 0.64–0.76 or 0.44–0.54 more lambs than those with genotype AA, respectively. These results preliminarily showed that the prolactin receptor locus is either a major gene that influences the prolificacy in Small Tail Han sheep or is in close linkage with such a gene.     

The effect of age on the morphometric sperm traits of domestic pigs (Sus scrofa domestica)

The experiment was conducted using 20 male domestic pigs, which were in 2 (equal-sized) age groups: under 14 months old and over 18 months old. At least 5 ejaculates from each male were taken, and in each ejaculate, morphometric measurements of 50 spermatozoa were made. The measured parameters were head area, head length and width, and flagellum length. For each ejaculate, the basic physical traits and the frequency of occurrence of developmental anomalies of the spermatozoa were examined. It was found that sperm dimensions tended to increase along with the boars' age. Considerable and statistical differences in head area and flagellum length were proved. Spermatozoa collected from older boars (above 18 months of age) had a head area larger by 0.49 microm(2)(P< or = 0.01) and a flagellum longer by 0.67 microm (P< or =0.01) than spermatozoa collected from younger boars (under 14 months of age). The differences in head length and width between the spermatozoa of the tested boars were considerably smaller and were not statistically provable. Significant correlations between the head area of spermatozoa and the head length (r = 0.56, P< or =0.05) and head width (r = 0.36, P< or =0.05) were found. However, the head length was not significantly directly correlated with its width (r = 0.18, P< or =0.05), and flagellum length was negatively correlated with spermatozoan head width (r = -0.71, P< or =0.05). Slight correlations between the morphometric traits of the sperm and the physical traits of the ejaculates (r = -0.11 to 0.16) were found, although in most cases, the correlations were not statistically provable.     

Effects of a high semen-collection frequency on the quality of sperm from ejaculates and from six epididymal regions in boars

This study examines the effect of semen-collection rhythm on the sperm maturation process in boar epididymis. Three post-pubertal boars were submitted to a high semen-collection frequency (stressed boars) during 4 days, and three males were kept as a control group (control boars). Semen samples coming from six epididymal regions and from the ejaculate of each male were evaluated for sperm concentration, sperm vitality, sperm motility and sperm morphology. In each epididymal region, either fluid resorption or fluid secretion was determined from the variation in sperm concentration. The pattern of fluid resorption-secretion along the epididymal duct differed significantly between the stressed and control boars. A high semen-collection frequency also affected the development of sperm motility and the sperm cytoplasmic droplet displacement along the epididymal duct. The incidence of some sperm abnormalities was also found to be higher in some epididymal regions and ejaculates of stressed boars. From the results of this study, it can be concluded that a high semen-collection frequency brings about an altered resorption and secretion pattern of the epididymal fluid, which results in defective sperm maturation and abnormal development of sperm motility.     

Prolactin receptor as a candidate gene for prolificacy of small tail han sheep

DNA polymorphism of the ovine prolactin receptor gene (PRLR) was investigated and used to study its effect on litter size in sheep. By means of PRLR gene sequence homology between sheep and human, three primer pairs were designed for polymerase chain reaction (PCR) amplification within intron 1 and exon 10 of the PRLR gene in sheep. In these parts of the gene the single nucleotide polymorphisms were detected by PCR-single strand conformation polymorphism (SSCP) in 314 Small Tail Han ewes. These poly-morphisms were used to study the associations with litter size. The results indicated that there were three genotypes (AA, AB and BB) detected by three primer pairs. For three primer pairs the frequency of allele A was 0.96, 0.79, 0.68; and the frequency of allele B was 0.04, 0.21, 0.32, respectively. The frequency of genotype AA was 0.93, 0.62, 0.51; the frequency of genotype AB was 0.06, 0.34, 0.34; the frequency of genotype BB was 0.01, 0.04, 0.15, respectively. The Small Tail Han ewes with genotype BB or AB had 0.64-0.76 or 0.44-0.54 more lambs than those with genotype AA, respectively. These results preliminarily showed that the prolactin receptor locus is either a major gene that influences the prolificacy in Small Tail Han sheep or is in close linkage with such a gene.     

The effects of postnatal FSH substitution on Sertoli cell number and the sperm production capacity of the adult boar

The role of FSH for Sertoli cell establishment and sperm production in the boar is not definitely known. In order to elucidate its function FSH was substituted postnatally in male pigs and the resulting effects on testicular histological traits and sperm production capacity were investigated when the boars had reached maturity. Six male piglets received pFSH from 18 to 48 days postnatally. Another six piglets instead received saline and served as controls. Blood samples were drawn to measure FSH, LH, testosterone and estradiol. After 28 weeks, the boars were trained to mount a dummy so that the spermatogenic capacity was tested by increasing the frequency of semen collection at the age of 30 weeks. Libido (latency time) and ejaculate criteria (volume, motility, morphological abnormalities) were determined. Thereafter the boars were killed and their testes analyzed for morphology, number of Sertoli cells, germ cells and Leydig cells as well as the ratio between mitosis and apoptosis in the tubules.FSH concentrations were twofold due to FSH application when compared to the controls. LH was low during the first 2 weeks of FSH treatment. Thereafter concentrations increased in three of the six treated animals but not in controls. Testosterone increased slightly over the application period both in the controls and the treated piglets. Estradiol levels were similar in both groups. Increased ejaculation frequency reduced sperm concentrations and sperm motility in all boars and the percentage of morphologically abnormal sperm increased. Ejaculate volumes and the time of latency were not significantly altered. No differences were observed between the controls and the FSH treated boars. The testicular parameters of both FSH- and control boars were identical for morphology, number of spermatogenic and somatic cells as well as mitosis–apoptosis equilibrium. The data demonstrated that a prolonged postnatal period of FSH concentrations does not influence the sperm production of the adult boar.     

Genetic and phenotypic variation in reproductive traits of AI boars

The primary objective of this paper is to review our current understanding of phenotypic variation in reproductive traits of AI boars. The proportion of boars that cannot be trained for collection in commercial studs is low and differences among genetic lines are small. In contrast, there is a considerable variation in sperm production and significant differences are present among genotypes. The general pattern is for sperm numbers to increase rapidly between 9 and 13 months of age and then gradually reach a plateau. This initial period of enhanced production occurs over a longer period in some genetic lines, resulting in differences of 30 x 10(9) sperm cells or more per ejaculate. There also are genetic lines of boars that seem to have a high "heat tolerance". Decreases in sperm production during periods of high environmental temperatures average 5-7% in these lines, compared with 15-20% in others. Finally, there are boars currently being used in the industry that are capable of producing exceptional fertility results with low numbers of sperm. Unfortunately, several breeding practices common to swine AI make their routine identification difficult. Based on the phenotypic variation observed in modern terminal sire lines of AI boars, current prospects for influencing sperm production, boar fertility, and mounting behaviours through genetic selection are viewed as being good, moderate to low, and poor, respectively.     

Characterization of the semen quality of postpuberal boars with spontaneous unilateral abdominal cryptorchidism on the right side.

In recent studies, we found that the ectopic testis from postpuberal boars with unilateral abdominal cryptorchidism does not produce sperm. Therefore, in these males, the seminal characteristics can be used as indicators of the activity of the scrotal testis and its epididymis and also the accessory glands. The semen quality (ejaculate volume, cell-rich fraction volume, sperm concentration, sperm vitality, sperm motility, sperm morphology and cephalic stability of spermatozoa) was evaluated in healthy postpuberal boars and in postpuberal boars with unilateral abdominal cryptorchidism on the right side. In comparison with the healthy boars, the unilateral abdominal cryptorchid boars showed a significant decrease of the ejaculate volume, sperm concentration and sperm motility. The low sperm concentration indicated that unilateral abdominal cryptorchidism severely impairs the sperm production of the scrotal testis. The decrease of ejaculate volume was attributed to an abnormal activity of the accessory glands. The alterations in sperm motility develop as a result of dysfunctions in the epididymal epithelium and/or the accessory glands. The sperm vitality, sperm morphology and cephalic stability of spermatozoa maintained normal values; therefore, at testicular level, despite the low sperm production, the germ cell differentiation is not disturbed. At epididymal level, the morphological maturation of spermatozoa is not altered.     

Sperm competition, sperm numbers and sperm quality in muroid rodents

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An "overall sperm quality" parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert.     

藏猪繁殖性状多基因效应分析

藏猪是我国特有的高原型地方猪种, 具有适应高海拔恶劣气候环境、抗病、耐粗、肉质鲜美等特点, 但繁殖力低。文章以促卵泡素b亚基(FSHb)、雌激素受体(ESR)、促乳素受体(PRLR)和视黄醛结合蛋白4(RBP4)等基因作为繁殖性状主要候选基因, 测定76头藏猪的基因型, 分析单基因和多基因合并对繁殖性状的影响效应。结果说明, 在藏猪群体中, FSHb、ESR和PRLR基因不同基因型繁殖性状差异显著, 优良基因型分别为BB、BB和AA。藏猪RBP4基因只有2种基因型, 对繁殖性状影响不显著。FSHb-ESR-PRLR基因的合并优良基因型为BB-BB-AA, 多基因聚合效应高于单基因效应。     

Intraspecific variation in ejaculate traits of the northern watersnake (Nerodia sipedon)

Sperm competition occurs when the sperm of more than one male compete to fertilize the eggs of a female. In reptiles, sperm competition is particularly prevalent and is an important agent of sexual selection in males. Spermatogenesis in reptiles can be energetically expensive, suggesting that there may be costs to producing high-quality ejaculates. The northern watersnake Nerodia sipedon has a mating system characterized by aggregations in which a single female mates with multiple males, resulting in high levels of multiple paternities. Under these circumstances sperm competition is likely important, and selection should favour sperm and ejaculate traits that enhance a male's reproductive success. In this study, we examined intraspecific variation in ejaculate quality (sperm length, motility, sperm density, spermactocrit) in male watersnakes and determined whether ejaculate traits varied with body size and condition, using both size-corrected mass and haematocrit as indices of condition. We found large variation among males in all these traits, except for sperm length. Although there was significant variation in sperm length among males, the majority of variation in sperm length occurred within rather than among individuals. Males with high haematocrit had sperm that were less variable with respect to length, and large males produced ejaculates that were less concentrated with respect to sperm than small males. The lack of condition dependence of most ejaculate traits is consistent with previous studies that indicate that male reproductive effort in this species is generally not energy limited, perhaps because of opportunistic foraging during the mating season.     

B-mode ultrasonographic evaluation of paired testicular diameter of mature boars in relation to average total sperm numbers

Crossbred, meat-type terminal sire boars (n = 215) were randomly assigned by age group (240-300, 301-360, 361-420, 421-480, 481-540, 541-600, and >721 days). Stud boars were on a once or twice weekly semen collection schedule. Testis diameters, in duplicate, were obtained using B-mode ultrasonography. Summation of average left and right testis diameter within boar gave the paired testicular diameter (PTD). Average ejaculate volume, sperm concentration (sperm/ml), and total sperm numbers for each boar were determined using composite data (average values) obtained from the last four semen collections. There was a <0.5cm difference between left and right testis diameters, with the left testis being the larger of the two testes (P = 0.03). There was no difference in PTD found between age groups in this study. Conversely, a dramatic increase in average total sperm numbers (ATSN) was observed between boars of 240-300 days (57.0+/-27.4 x 10(9) sperm) and up to 420 days (118.6+/-33.6 x 10(9) sperm) of age. The ATSN (127+/-32.5 x 10(9) sperm) remained constant for the 421-480 to >721-day age groups. The correlation between PTD and ATSN was low (r = 0.24) in this study. The results of this study demonstrate that normal boars should exhibit <0.5cm diameter difference between testes. As observed in other studies, the left testis was usually larger than the right testis. Correlation of total sperm numbers in a boar ejaculate using a composite ejaculate score (average values) and PTD measurements obtained using B-mode ultrasonography was poor when used in boars >8 months of age.     

藏猪3个繁殖性状主效基因多态性研究

藏猪是我国高海拔特有猪种,为了解藏猪繁殖性能的遗传潜力和基因多样性,利用PCR－RFLP技术测定了202头藏猪ESR、FSHβ和PRLR 3个繁殖性状主效基因位点的基因型多态性,并与文献报道的国内外猪种进行比较。结果显示藏猪群体ESR、FSHβ和PRLR优势基因型分别为BB型、AB型和AA型,3个位点基因型分布均符合哈迪－温伯格平衡。藏猪群体3个繁殖性状主效基因位点都有报道的有利基因型,且有利等位基因频率较高,说明藏猪在这3个基因位点可能具有优良繁殖性能的遗传潜力。     

The effect of a c.-8G>T polymorphism on the expression of cytochrome b5A and boar taint in pigs

The level of cytochrome b5A ( CYB5A ) in pig testis is correlated with boar taint from androstenone and an AF016388:c.-8G>T polymorphism in CYB5A has been linked with low androstenone levels in the fat of pigs. In this study, we developed a polymerase chain reaction-based assay to genotype 1242 boars from eight lines for the c.-8G>T SNP. The c.-8T allele was found in all eight lines at a frequency ranging from 1.8% to 20.3% with an overall frequency of 8.6%. Significant deviations from Hardy–Weinberg equilibrium were found in the Hampshire, Landrace and Yorkshire breeds. The homozygous mutant c.-8TT occurred infrequently and was not found in some lines, but was consistently associated with low androstenone levels in fat. Both CYB5A mRNA and CYB5A protein levels were decreased in the c.-8TT genotype in a subset of Yorkshire boars, suggesting that low levels of CYB5A protein in the c.-8TT mutant were not due to inefficient translation of CYB5A mRNA. There were significant but modest marker effects on fat androstenone levels in Landrace, Yorkshire and a Large White/Duroc cross and fat skatole in Duroc and Sire Line breeds. There was no effect of CYB5A genotype on bulbourethral gland length, suggesting that this SNP will not affect reproductive traits. We conclude that the c.-8G>T SNP in the CYB5A gene has a significant but modest effect on boar taint in male pigs and could be useful in some breeds as part of a panel of SNP markers in a marker-assisted selection programme to produce low boar taint pigs.     

The frequency of multiple paternity predicts variation in testes size among island populations of house mice

Polyandry generates selection on males through sperm competition, which has broad implications for the evolution of ejaculates and male reproductive anatomy. Comparative analyses across species and competitive mating trials within species have suggested that sperm competition can influence the evolution of testes size, sperm production and sperm form and function. Surprisingly, the intraspecific approach of comparing among population variation for investigating the selective potential of sperm competition has rarely been explored. We sampled seven island populations of house mice and determined the frequency of multiple paternity within each population. Applying the frequency of multiple paternity as an index of the risk of sperm competition, we looked for selective responses in male reproductive traits. We found that the risk of sperm competition predicted testes size across the seven island populations of house mice. However, variation in sperm traits was not explained by sperm competition risk. We discuss these findings in relation to sperm competition theory, and other intrinsic and extrinsic factors that might influence ejaculate quality.