Detection of Sex Chromosome Aneuploidy in Equine Spermatozoa Using Fluorescence In Situ Hybridization

The aim of our study was to diagnose aneuploidy in equine spermatozoa by multicolour fluorescence in situ hybridization (FISH) technique using specific molecular probes for equine sex chromosomes and autosome pair four (EGFR probe) labeled by different fluorochromes. These were applied on decondensed spermatozoa of four stallions. In total, more than 8800 sperm cells were examined. The total frequency of aberrant cells was 0.496%: aneuploidy of XX (0.135%), YY (0.023%), XY (0.102%), diploidy (0.057%), lack of sex chromosome (0.18%). In one stallion the ratio of normal X‐ and Y‐bearing cells was different from the expected 1 : 1 ratio (p = 0.0002), in all three other stallions this ratio was close to 1 : 1. The present study demonstrated that the FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and allows for the determination of the ratio between X–Y‐spermatozoa.     

Short‐Term Storage and Swim‐Up Selection Do Not Affect the X/Y Ratio in Equine Spermatozoa

The standard procedure of artificial insemination with fresh equine spermatozoa involves short‐term storage (to 48 h at 5°C). This procedure is accompanied by a gradual loss of sperm viability. The aim of this study was to investigate whether the X/Y ratio of equine spermatozoa is affected by short‐term storage and the swim‐up procedure. We used a standard protocol, for short‐term storage (0, 24 and 48 h at 5°C) of stallion semen diluted in the commercial extender EquiPro? (Minitüb GmbH, Tiefenbach, Germany). After each set‐up storage period, the motile fraction of sperm cells was selected by the swim‐up method. The X/Y ratio was evaluated by fluorescence in situ hybridization (FISH) in the fresh, non‐selected sperm, and in motile spermatozoa selected after each of the storage periods. Molecular probes for the equine chromosomes X and Y were used. The X/Y ratio in all sperm samples analysed in this study (fresh and stored) was not different from the theoretical 1 : 1 value. The incidence of chromosomally abnormal sperm cells in the fresh (0.28%) and motile (0.13%) sperm samples was not significantly different. The two approaches (sperm storage up to 48 h and the swim‐up procedure) applied to this study did not affect the X/Y ratio in the motile fraction of equine spermatozoa. This finding does not conform to phenomena described for human and cattle. For this reason, the finding may imply species‐related differences.     

Enrichment of Y‐Chromosome‐Bearing Bull Spermatozoa by Swim‐up Through a Column

With the advancement of assisted reproductive biotechnologies, preselecting the sex of offspring has become an important goal for cattle and other livestock breeding as well as for research. The aim of this study was to investigate the feasibility of producing enriched pools of X‐ or Y‐chromosome‐bearing sperm by vertical swim‐up through a long, narrow column. Sperm recovered from the top portion of the column was predominantly Y‐bearing (60%, p < 0.05), which were capable of fertilizing matured oocytes and produce significantly more male embryos compared with standard swim‐up protocol.     

水牛精液中X和Y精子比率的鉴定

利用流式细胞仪分析水牛分离和未分离精液中精子的DNA含量,所得的直方图用高斯曲线拟合,分析计算出样本中X和Y精子的比率。结果表明:未分离的水牛精液中X精子的比率为50%,与正常水牛后代性别比率没有显著差异;而水牛的分离X精液样本中X精子占93%,分离Y精液样本中Y精子占89%。实验结果显示了流式细胞仪DNA分析法鉴定水牛精液中X和Y精子比率的可靠性,而流式细胞仪分离精子程序和方法在水牛上的应用是可靠而有效的。     

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted,frozen–thawed spermatozoa in field conditions

The aim of this study was to evaluate fertility and sex ratios after artificial insemination in dogs under field conditions. Semen was cryopreserved as unsorted (control) or was separated into X‐ and Y‐chromosome‐bearing sperm using a cell sorter. Sixty female dogs were inseminated with frozen–thawed spermatozoa of 100 × 10⁶ unsorted (a dose in practice) and 4 × 10⁶ sorted (X and Y group, respectively). A total of 20 dogs became pregnant and 126 puppies were born from the three groups. The percentage of parturition was similar for the X (5/20; 25.0%) and Y (4/20; 20.0%) group (P > 0.05), but lower than controls (11/20; 55.0%) (P < 0.05). Ultimately 28 out of the 32 puppies produced from X group were female (87.5%) and 19/22 (86.4%) puppies of Y group were male. In contrast, sex ratio (51.4% to 48.6%) in the control was significantly different from the X, Y group (P < 0.05). However, male and female puppies in the control had similar birth weights and weaning weights to those from the X and Y groups. This preliminary information indicated that normal puppies of predicted sex can be produced with low numbers of sorted cryopreserved dog spermatozoa at a farm level, making sperm‐sexing technology potentially applicable for elite breeding units.     

Quantification of the DNA Difference,and Separation of X‐ and Y‐Bearing Sperm in Alpacas (Vicugna pacos)

Sperm sexing is an emerging reproductive technology which has been successfully used to produce offspring of a pre‐determined sex in domestic and wildlife species but has yet to be applied to New World camelids. The aims of the present study were to (i) optimize the Hoescht 33342 (H33342) staining concentration for the flow cytometric separation of X and Y chromosome‐bearing alpaca (Vicugna pacos) sperm nuclei, (ii) separate alpaca sperm nuclei into high purity (>90%) populations bearing the X‐ and Y‐chromosome and (iii) determine the DNA difference between X‐ and Y‐bearing sperm in alpacas. Semen was collected from alpacas and sperm nuclei stained with H33342, incubated and analysed using a high‐speed cell sorter (SX‐MoFlo^®). H33342 staining concentrations of 36, 54, 72 or 90 μm did not affect the proportion of correctly oriented sperm nuclei (43.3 ± 3.9, 46.4 ± 3.7, 44.5 ± 4.0 and 51.1 ± 2.5% respectively) nor the speed of sorting (1381 ± 160, 1386 ± 123, 1371 ± 133 and 1379 ± 127 sperm nuclei/s). Sort reanalysis determined high levels of purity for X‐ and Y‐enriched populations (96.6 ± 0.7% and 96.1 ± 1.1% respectively). The DNA difference, based on fluorescence intensity (determined by the SX‐MoFlo^®), was 3.8 ± 0.06%. These data demonstrate for the first time that alpaca sperm nuclei can be separated into high purity populations and the potential for applying sperm sexing technology to New World camelids.     

The application of genetic markers for cell chimerism diagnosis in lambs

A total of 181 Romanov lambs, including 84 pairs of twins, three litters of triplets and one litter of quadruplets, were investigated. Erythrocyte antigens belonging to six blood group systems were determined with 16 reagents. Six microsatellite loci BMS360, INRA123, McM42, CSSM66, ETH225 and TGLA53 were used to genotype the lambs with automated DNA sizing technology. For cytogenetic diagnosis of leucocyte chimerism, the fluorescence in situ hybridization (FISH) was applied using bovine X and Y chromosome painting probes. The blood‐group typing of lambs from twin and multiple pregnancies allowed us to detect nine cases of erythrocyte chimerism in the investigated population. The analysis of microsatellite DNA sequences showed the presence of cell chimerism in 13 animals. The hybridization of a bovine X and Y chromosome‐specific probe resulted in two yellow brightly luminescent signals in 54,XX cells, and one yellow and one blue signal in 54,XY cells helped us diagnose chromosomal chimerism in seven animals.     

Disorder of sex development in a cat with chromosome mosaicism 37,X/38,X,r(Y)

An 18‐month‐old European shorthair cat was subjected to genetic studies due to ambiguous external genitalia (underdeveloped both penis and scrotum). Further anatomic and histopathological studies revealed the presence of abdominal, atrophic testes and uterus. Cytogenetic analysis showed two cell lines, one with X monosomy—37,X [90% of the analysed metaphase spreads], and other line had 38 chromosomes with normal X chromosome and abnormally small Y‐derived chromosome—38,X,der(Y) [10%]. Further fluorescence in situ hybridization study with telomeric probe revealed a ring structure of the der(Y). Eight Y chromosome‐specific genes, SRY, TETY1, TETY2, CUL4BY, CYORF15, HSFY, FLJ36031Y and ZFY, were detected. We conclude that the described abnormality of the reproductive system, leading to sterility, was caused by a very rare type of chromosomal mosaicism—37,X/38,X,r(Y).     

野牦牛mtDNA Cytb基因全序列测定及系统进化关系

为从分子水平探究牦牛的分类地位和遗传多样性,试验测定了野牦牛细胞色素b基因全序列,并以绵羊为外群,构建野牦牛、家牦牛、大额牛、普通牛、瘤牛、水牛、非洲野牛、欧洲野牛、美洲野牛、非洲水牛等牛亚科种间系统进化树.结果表明:野牦牛细胞色素b基因全序列长1 140bp,序列间共有13个SNP多态位点,核苷酸变异类型包括转换和颠换,无插入和缺失,表明野牦牛具有较丰富的遗传多样性.研究结果支持将牦牛划分为牛亚科中一个独立属(即牦牛属)的观点.     

Milk protein polymorphisms in cattle (Bos indicus), mithun (Bos frontalis) and yak (Bos grunniens) breeds and their hybrids indigenous to Bhutan

In the current study, milk protein variation was examined in cattle (Bos indicus), mithun (Bos frontalis), yak (Bos grunniens) and their hybrid populations in Bhutan to estimate genetic variability, conduct genetic characterization and assess the possibility of gene flow between mithun and cattle. Isoelectric focusing of 372 milk samples from 11 populations detected four molecular types of β‐lactoglobulin (A, B, E and M), five molecular types of α_S1‐casein (A, B, C, E and X) and three molecular types of k‐casein (A, B and X). Mithun and yak shared alleles but were found to exhibit different allele frequencies for the proteins studied. The degree of genetic variability within populations was measured by average heterozygosity and ranged from 24–40% in cattle, 26% for yak and 33% for mithun. We also resolved the traditional mithun and cattle hybridization system via principal component analysis. Our results suggested secondary introgression of mithun genes to the village Thrabum population, and a close genetic relationship between Bhutanese indigenous cattle and Indian cattle.     

不同物种Y染色体特异基因产物的研究

根据牛Y染色体3个特异基因设计3对引物，分别对牛、牦牛、绵羊、山羊、猪、小鼠基因组进行PCR扩增。3对引物在牛和牦牛基因组中都得到了扩增产物，在绵羊的扩增中只检测到了1个性别决定基因的扩增产物，在其他物种中均没有检测到扩增产物。将其扩增产物进行克隆测序，并采用DNAMAN软件对测定序列进行比对分析，结果表明：3对引物的扩增结果在牦牛和牛的同源性分别达到了99％以上，性别决定基因的扩增产物在绵羊和牛间的同源性达到了94％。     

Recovery of sperms bearing X chromosomes with different concentrations of magnetic nanoparticles in ram

Pre-conceptual sex selection is still a highly debatable process whereby X and Y chromosome bearing spermatozoa are isolated before oocyte fertilization. Recently, magnetic nanoparticles (MNP) have been used to determine X and Y chromosomes bearing spermatozoa as a result of searching for a cheap, highly efficient method using non-toxic materials. This study aimed to recover the sperm bearing X chromosomes in ram with different concentrations of MNP and then evaluate the success of this method using polymerase chain reaction (PCR). Ram sperms were divided into four groups, treated with 0 (control), 50, 100 and 200 μg/ml MNP, respectively. MNP was used to restore sperm cells bearing X chromosomes. Upon recovery, the PCR was performed to identify the X and Y sperms, Methyl ThiazoleTetrazolium (MTT), to assess MNP toxicity and sperm viability and acridine orange (AO) to evaluate sperm DNA integrity. The results of PCR revealed that the treatment of spermatozoa- bearing X chromosomes with 50 μg/ml MNP had the highest effects on the recovery of X sperm rather than the other concentrations of MNP. However, the concentrations of MNP did not have any toxic effects on spermatozoa, sperm viability and, DNA integrity, but the high concentration of MNP (200 μg/ml) significantly reduced DNA integrity. According to MTT and AO results, the concentrations of MNP used in this study had no toxic effects on spermatozoa and did not reduce the sperm viability and DNA integrity, except that 200 μg/ml MNP significantly reduced DNA integrity.     

牦牛和普通牛种间杂种公牛睾丸的组织学观测与研究

通过对牦牛和黄牛种间杂种公牛及未成年公牦掉睾丸的比较组织学观测，结果表明：杂种牛雄性不育的生殖隔离机制与生精小管及生精上皮细胞的发育状况、生精小管基膜的厚度、生精小管的间距、间质组织细胞的发育状况、间质组织与生精小管间形成大量组织液等因素有关。随种间杂交代数增加，睾丸组织的发育状况逐渐改善，精原细胞的死亡率相应降低，杂种3代（F3）已能完成减数分裂产生精子，并显示出精原细胞的死亡率的逐代减少与父本基因含量逐代增多有一定的相关性。     

Comparative proteomic analysis of high‐ and low‐fertile buffalo bull spermatozoa for identification of fertility‐associated proteins

The present study identified few potential proteins in the spermatozoa of buffalo bulls that can be used as an aid in fertility determination through comparative proteomics. The sperm proteome of high‐fertile buffalo bulls was compared with that of low‐fertile buffalo bulls using two‐dimensional difference gel electrophoresis (2D‐DIGE), and the differentially expressed proteins were identified through mass spectrometric method. The protein interaction network and the functional bioinformatics analysis of differentially expressed proteins were also carried out. In the spermatozoa of high‐fertile bulls, 10 proteins were found overexpressed and 15 proteins were underexpressed at the level of twofold or more (p ≤ 0.05). The proteins overexpressed in high‐fertile spermatozoa were PDZD8, GTF2F2, ZNF397, KIZ, LOH12CR1, ACRBP, PRSS37, CYP11B2, F13A1 and SPO11, whereas those overexpressed in low‐fertile spermatozoa were MT1A, ATP5F1, CS, TCRB, PRODH2, HARS, IDH3A, SRPK3, Uncharacterized protein C9orf9 homolog isoform X4, TUBB2B, GPR4, PMP2, CTSL1, TPPP2 and EGFL6. The differential expression ranged from 2.0‐ to 6.1‐fold between the two groups, where CYP11B2 was high abundant in high‐fertile spermatozoa and MT1A was highly abundant in low‐fertile spermatozoa. Most of the proteins overexpressed in low‐fertile spermatozoa were related to energy metabolism and capacitation factors, pointing out the possible role of pre‐mature capacitation and cryo‐damages in reducing the fertility of cryopreserved buffalo spermatozoa.     

Differences in genetic structure assessed using Y‐chromosome and mitochondrial DNA markers do not shape the contributions to diversity in African sires

Up to 173 African sires belonging to 11 different subpopulations representative of four cattle groups were analysed for six Y‐specific microsatellite loci and a mitochondrial DNA fragment. Differences in Y‐chromosome and mtDNA haplotype structuring were assessed. In addition, the effect of such structuring on contributions to total genetic diversity was assessed. Thirty‐five Y‐chromosome and 71 mtDNA haplotypes were identified. Most Y‐chromosomes analysed (73.4%) were of zebu origin (11 haplotypes). Twenty‐two Y‐haplotypes (44 samples) belonged to the African taurine subfamily Y2a. All mtDNA haplotypes belonged to the “African” taurine T1 haplogroup with 16 samples and nine haplotypes belonging to a recently identified subhaplogroup (T1e). Median‐joining networks showed that Y‐chromosome phylogenies were highly reticulated with clear separation between zebu and taurine clusters. Mitochondrial haplotypes showed a clear star‐like shape with small number of mutations separating haplotypes. Mitochondrial‐based F_ST‐statistics computed between cattle groups tended to be statistically non‐significant (p > .05). Most F_ST values computed among groups and subpopulations using Y‐chromosome markers were statistically significant. AMOVA confirmed that divergence between cattle groups was only significant for Y‐chromosome markers (Φ_CT = 0.209). At the mitochondrial level, African sires resembled an undifferentiated population with individuals explaining 94.3% of the total variance. Whatever the markers considered, the highest contributions to total Nei's gene diversity and allelic richness were found in West African cattle. Genetic structuring had no effect on patterns of contributions to diversity.     

牦牛肌红蛋白的基因克隆测序、分离纯化、含量及其与乳酸脱氢酶和苹果酸脱氢酶活力的关系

为探索牦牛适应高原缺氧环境的分子机制,测定了麦洼牦牛心肌和骨骼肌中肌红蛋白（Mb）含量及乳酸脱氢酶（LDH）、苹果酸脱氢酶（MDH）活力,并对牦牛Mb进行了分离纯化和基因克隆测序。牦牛心肌和骨骼肌中Mb含量显著高于水牛和黄牛（P〈0.01）;牦牛、黄牛和水牛心肌MDH/LDH活力比均显著高于骨骼肌;牦牛心肌MDH/LDH活力比显著高于水牛和黄牛,但在不同牛种骨骼肌之间无显著差异。各组织Mb含量与MDH/LDH活力比呈显著正相关。试验用RT-PCR方法从牦牛心肌中克隆了Mb基因,与普通牛相比,核苷酸序列同源性为99.5%,氨基酸序列完全相同;用盐析、CM-Sephadex阳离子交换层析、Sephadex G-50凝胶层析等方法分离纯化了牦牛Mb,SDS-PAGE显示其分子量约为17 ku。     

杂种黄公牛与牦牛杂交的F_1代生长发育测定

揭示了(西×黄)♂×牦牛♀和(黑×黄)♂×牦牛♀两个牦牛三元杂交组合繁殖的F1代犏牛生长发育情况,结果表明,F1代犏牛各年龄段的体尺、体重杂种优势明显,且母犏牛提前1～2岁投入生产,建议在牦牛杂交改良中推广这两个牦牛三元杂交组合。     

麦洼牦牛SRY基因克隆及序列分析

SRY是Y染色体上具体决定生物雄性性别的基因片段,是多数哺乳动物性别决定基因之一。本试验采用克隆测序SRY基因并结合生物信息学对其序列进行分析,利用软件Codon W分析麦洼牦牛、普通牛、绵羊、山羊、猪、小鼠、鸡和人该基因编码区的密码子偏好性。克隆测序得到SRY基因序列含1个690 bp的开放阅读框,共编码229个氨基酸;其编码区核苷酸序列与普通牛、绵羊、山羊、猪、小鼠、鸡、人相应序列间的一致性分别为99.9%、93.9%、91.2%、76.5%、43.3%、21.4%、69.1%。经聚类分析,麦洼牦牛首先与普通牛聚在一起,绵羊与山羊聚为一类,这两类相聚后再依次与猪、人、小鼠相聚,最后与鸡聚为一类,其结果与以往的生物学分类结果一致。该基因编码的蛋白质分子式为C₁₁₅₅H₁₈₂₇N₃₅₁O₃₄₈S₁₁,相对分子质量为2655.0,理论等电点PI为9.55,亲水性平均数为-0.855。其密码子偏好性分析显示,T3_s为0.2905、C3_s为0.3240、A3_s为0.3728、G3_s为0.2963,第3位碱基中出现G和C占第3位碱基总量的48%,同义密码子数为61。上述数据表明麦洼牦牛SRY基因编码序列与普通牛、绵羊、山羊、猪、人具有较高的一致性,在物种进化过程中较为保守。该基因对含A的密码子有较高的偏爱性,尽量避开以G结尾的密码子,且其所编码的蛋白质为亲水性蛋白。