人γ－精浆蛋白的亲和纯化及其糖基化分析

目的:从人精液中提纯γ-精浆蛋白并对其生物学特性进行鉴定。 方法:应用饱和硫酸铵法从小鼠腹水中纯化出抗γ-精浆蛋白的单克隆抗体E4B7,将其共价交联到CNBr-Sepharose4B上,制备成免疫亲和层析柱,用该层析柱亲和纯化经饱和硫酸铵粗提的精液标本。纯化产物分别用SDS-PAGE、Western-blot及ELISA进行生物学特性鉴定,最后经PNGase F、Endo-H酶消化后进行糖基化分析。结果:SDS-PAGE电泳表明纯化蛋白的相对分子量约为44KD,Western-blot及ELISA鉴定显示该蛋白可与抗γ-精浆蛋白的单抗E4B7特异性结合。对纯化蛋白无论是单用Endo-H酶消化,还是同时用Endo-H酶和PNGase F酶共同消化,消化产物电泳后相对分子量均降低到34KD左右,而单独用PNGase F酶消化后相对分子量没有改变。Western-blot及ELISA鉴定显示糖苷酶处理后的纯化蛋白仍可与单抗E4B7特异性结合。结论:成功纯化出N-糖基化形式的γ-精浆蛋白,这为下一步筛选人源化抗体奠定了纯的抗原基础。     

不经染色直接从SDS-PAGE制备凝胶中准确切下所需蛋白条带的方法

蛋白样品的垂直板SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE)不但是一种最常用的蛋白分析方法,也经常用于蛋白质的制备。从电泳凝胶上纯化蛋白,一般都要先用考马斯亮蓝染色,然后切下所需的蛋白条带。这里介绍一种可以不染色,直接从SDS-PAGE制备凝胶上准确切割所需蛋白条带的方法。与染色后切胶的方法相比,这种方法简单、省时,分离到的蛋白容易从胶中洗脱回收,并可明显提高回收率,而且省去了令人烦怖的从回收的蛋白中脱去染色时结合的染料的问题。作者曾用此方法分离过多种蛋白,屡试不爽。这种方法与一般的SDS-PAGE制备电泳的差别主要在电泳结束后对凝胶的处理上。     

圈养大熊猫冷冻精液对其种群遗传多样性的作用

在过去34年的圈养大熊猫种群保护工作中,我们成功建立了全球最大的大熊猫精子库,目前已保存50只大熊猫个体总计7 000余支细管冷冻精液(冻精)。冷冻精液一方面可以使物种的遗传资源得到长久保存,另一方面可以通过人工授精的方式促进种群繁育。但是,圈养大熊猫冷冻精液对其种群遗传多样性的作用尚未有明确报道。本研究首先根据成都大熊猫繁育研究基地2000—2014年冷冻精液人工授精数据,对比分析了冻精人工授精个体和圈养种群的遗传多样性。结果显示,冻精人工授精个体遗传多样性均高于同年圈养种群的平均遗传多样性,表明在繁殖年份中冻精人工授精可以显著提高圈养大熊猫种群的遗传多样性。统计精子库中所有冻精个体的平均血缘系数并与圈养种群进行对比分析,探究冷冻精液对圈养种群遗传多样性的潜在作用。结果显示,精子库中有21只已死亡个体的精液,其中有66.67%的个体平均血缘系数低于圈养种群;有14只20岁以上个体的精液,其中有50.00%的个体平均血缘系数低于圈养种群;另有15只20岁以下个体的精液,其中有53.33%的个体平均血缘系数低于圈养种群,表明冷冻精液对圈养种群遗传多样性的保护具有重要价值。综上所述,冷冻精液不但有效保存了大熊猫遗传资源,而且在保护圈养种群遗传多样性方面具有积极的促进作用。     

小菜蛾蛋白质提取方法的比较

建立适用于小菜蛾蛋白质组学研究的样品前处理方法。应用SDS-PAGE电泳比较分析了TCA-丙酮沉淀,Tris-饱和酚抽提,RIPA Lysis Buffer和I-PER Reagent直接裂解4种方法对小菜蛾幼虫总蛋白的抽提质量。结果显示:4种方法对小菜蛾全蛋白的提取率差异比较显著,其中RIPA Lysis Buffer和I-PER Reagent两种直接裂解法蛋白提取率最高,分别为52.06 mg/g和46.16 mg/g;TCA-丙酮沉淀法提取率适中,为34.58 mg/g;Tris-饱和酚抽提法提取率最低,为27.39 mg/g。SDS-PAGE电泳分析表明,Tris-饱和酚抽提法蛋白谱条带分辨率最高,蛋白条带最多,为32条,在17.0 k Da-245 k Da的范围内分布均匀;TCA-丙酮沉淀法蛋白谱带数目为30条,条带清晰,分辨率较高,但缺失部分蛋白;RIPA Lysis Buffer和I-PER Reagent两种直接裂解法蛋白谱条带数目较少,小分子蛋白条带丢失或不明显。根据蛋白提取率和条带的数量综合分析,TCA-丙酮沉淀法适用于小菜蛾蛋白质组学的样品前处理。     

苹果树腐烂病菌胞外蛋白提取方法

选择一种理想的蛋白提取方法,获得数量多、质量高的苹果树腐烂病菌Valsa mali胞外蛋白用于蛋白质组学分析,为全面解析该病原菌的致病机制奠定基础。采用冷冻干燥透析结合法、脱氧胆酸钠(DOC)-10%TCA沉淀法、硫酸铵沉淀法3种方法分别提取苹果树腐烂病菌的胞外蛋白,并筛选最适的硫酸铵浓度,利用Bradford法测定蛋白总量、SDS-PAGE检测蛋白条带分辨率及不同致病力菌株蛋白差异。结果表明,70%硫酸铵沉淀提取的胞外蛋白量最高、SDS-PAGE电泳可辨认蛋白条带最多。强弱致病菌株胞外蛋白的SDS-PAGE电泳图谱在37-50 kD有4条蛋白条带差异明显,由此表明,70%硫酸铵盐析法更适合用于苹果树腐烂病菌胞外蛋白质差异分析时蛋白质的提取。     

木本植物蛋白提取和SDS-PACE分析方法的比较和优化

在几种木本植物上对4种常用蛋白提取方法、4种凝胶染色和脱色方法及影响蛋白定量和SDS-PAGE电泳的因素进行了比较研究,发现：利用改良丙酮沉降法提取蛋白,不仅提取效率高,而且杂质干扰少,得到的电泳结果,蛋白条带清晰,数量多;将常规凝胶染色和脱色液中的乙醇改用为甲醇可大大改善染色和脱色效果,获得了没有背景或背景很浅的电泳结果。通过研究确定了一套优化的适用于木本植物的蛋白提取、定量、凝胶制备、电泳、染色、脱色以及干胶制备的方法。利用这一优化方法对胡杨细胞盐胁迫蛋白进行了研究,发现了与高水平盐胁迫有关的66kD蛋白和与盐胁迫和干旱胁迫均有关的28kD蛋白,获得了满意的蛋白SDS-PAGE分析结果。     

大肠杆菌glgC基因的克隆及原核表达

以大肠杆菌BL21染色体DNA为模板,根据glgC基因的全序列设计了1对引物,在优化的PCR反应条件下扩增出了glgC基因片段,测序结果显示该片段大小为1296 bp,编码432个氨基酸残基。将该基因克隆到原核表达载体pET-28a-c( )中,重组载体pET-glgC转化至大肠杆菌BL21(DE3),经IPTG诱导后,SDS-PAGE电泳鉴定,得到了与理论推算的glgC基因表达产物分子质量(约53 kD)相符的特异蛋白条带。     

人精子顶体膜蛋白的纯化及性质的研究

目的：提取和纯化与人卵透明带(HZP)作用更为密切的人精子膜蛋白,为改进精子包被抗原的制备方法和筛选避孕疫苗抗原提供科学依据。方法：将人精子膜蛋白混合液进行SDS-PAGE电泳,然后全胶电洗脱分剐收集蛋白溶液30管,以阳性管内的HZP特异精子蛋白溶液包被抗原进行AsAb检测。结果：与HZP结合的人精子膜蛋白有多种成分;全胶电洗脱后以第14管内成分之特异最强,其分子量范围为48～53KD。结论：以电洗脱转移法纯化筛选的人精子膜蛋白是特异的,与HZP具极强相关性,可以用该精子膜蛋白包被抗原进行AsAb研究。     

大熊猫脑钙调素的纯化及性质研究

以大熊猫脑为材料,经提取、热处理、Phenyl-Sepharose CL-4B疏水柱和快速液相分子筛层析,分离纯化得到CaM.经SDS-PAGE、 PAGE和IEF鉴定,得到的CaM为一条带.经测定,大熊猫脑CaM的分子质量为19 ku,等电点为3.8.酶活性实验表明大熊猫脑CaM对牛心磷酸二酯酶有激活作用.氨基酸组成分析结果与其他来源CaM相近.     

乙醇激活对小鼠透明带糖蛋白的生化修饰

为研究乙醇激活对小鼠卵透明带的修饰,用SDS-PAGE和HPLC分析未受精卵和乙醇激活卵的透明带糖蛋白。SDS-PAGE结果显示,未受精卵和乙醇激活卵的透明带糖蛋白成分的电泳带型相似。只是在还原电泳时,乙醇激活卵透明带样品中分离出一条约20kD的特异条带。未受精卵和乙醇激活卵的ZP3成分没有明显差异。HPLC结果显示：乙醇激活卵透明带有3个O-连单糖的洗脱峰,与未受精卵透明带的3个O-连单糖洗脱峰很相似;但是,乙醇激活卵透明带有另外两个O-连寡糖洗脱峰,RT值分别为12.09和8.89,未受精卵样品只有一个RT值为9.14的洗脱峰,他们代表一些O-连寡糖或寡糖混合物。可见,乙醇激活对ZP3多肽链和O-连单糖没有明显的影响。主要的修饰作用在O-连寡糖上。     

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of ram seminal plasma proteins and their correlation with semen characteristics

The present study was conducted to investigate fertility-associated proteins in ram seminal plasma and the correlation between specific protein and semen characteristics in sheep. Thirty-eight German merino sheep clinically proven healthy were chosen and divided into three groups according to fertility. Ejaculates were collected by an artificial vagina and semen characteristics (volume, pH value, motility, viability and concentration) were recorded. Seminal plasma was harvested by centrifugation and then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis in parallel with molecular weight standards. Fifteen protein bands with different molecular weights, ranging from 15.13 to 116.20 kDa, were identified on the gel. The results showed that the relative content of eight protein bands was significantly different between the high-fertility group (H-group) and the low-fertility group (L-group). Although the remaining seven protein bands showed no fertility-associated changes in their relative content, some of them were negatively or positively correlated with some semen quality parameters (motility, viability, concentration or pH value). Thus, this study indicates that ram seminal plasma contains specific proteins that are associated with fertility and semen characteristics. Also, these proteins could be utilised in developing a reliable and simple method to determine the ram fertility or semen quality.     

Characteristics of seminal plasma proteins and their correlation with canine semen analysis

The objectives were to separate canine seminal plasma proteins (with SDS-PAGE) and to determine the correlation between specific proteins and semen characteristics. Three ejaculates from 20 mixed-breed dogs, of unknown fertility, were collected by digital manipulation. Ejaculate volume and color, sperm motility, sperm vigor, percentage of morphologically normal spermatozoa, and membrane integrity (hypoosmotic swelling test and fluorescent staining) were assessed. For each dog, seminal plasma was pooled from all three ejaculates and proteins were separated with SDS-PAGE, using polyacrylamide concentrations of 13% and 22% in the separation gels. After staining, gel images were digitized to estimate molecular weights (MW) and integrated optical density (IOD) of each lane and of individual bands. Total seminal plasma protein concentration was 2.19+/-1.56 g/dL (mean+/-SD; range 1.12-5.19 g/dL). A total of 37 protein bands were identified (although no dog had all 37 bands). In the 13% gel, molecular weights ranged from 100.6 to 17.1 kDa, with four bands (49.7, 33.2, 26.4, and 19.5 kDa) present in samples from all dogs. In the 22% gel, molecular weights ranged from 15.6 to 3.6 kDa, with nine bands (15.6, 13.5, 12.7, 11.7, 10.5, 8.7, 7.8, 5.6, and 4.9 kDa) present in samples from all dogs. Combined for both gels, the majority of bands (85%) had molecular weights <17 kDa, with B20 (15.6 kDa) in high concentrations in samples from all dogs. There were positive correlations (P < or = 0.01) between two bands, B4 (67 kDa) and B5 (58.6 kDa), and sperm motility (r=0.66 and r=0.46), sperm vigor (r=0.56 and r=0.66), percentage of morphologically normal spermatozoa (r=0.55 and r=0.59), the hypoosmotic swelling test (r=0.76 and r=0.68), and fluorescent staining (r=0.56 and r=0.59), respectively. In conclusion, 37 proteins were identified in seminal plasma; two were significantly correlated with semen characteristics.     

SDS-PAGE characterization of the proteins in equine seminal plasma

The aims of this project were to document the protein profile of equine seminal plasma and determine the variability between stallions in the relative composition of proteins in the ejaculate. A single ejaculate was obtained from 14 stallions of varying breed and age. The gel fraction was removed by an in-line filter. The semen was centrifuged and the supernatant seminal plasma aspirated without disturbing the sperm pellet. The seminal plasma was recentrifuged and stored in cryovials at -70 degrees C. Samples were thawed, recentrifuged, assayed for protein concentration (BCA protein assay), divided into aliquots, then stored at -70 degrees C. A standard protein concentration of 50 microg was loaded in each 10 microl sample. SDS-PAGE was performed using 15% polyacrylamide and a mixture of molecular weight standards. The electrophoresed gel was stained for proteins with Coomassie blue, air-dried, then scanned by a megapixel camera interfaced to a computer. Image analysis software calculated integrated optical density (IOD) values for each lane, and bands within a lane. Each band IOD was expressed as a percentage of the total lane IOD, thus reflecting the relative concentration of each protein within the ejaculate. A total of 14 bands were identified, ranging from a large 120 kDa protein down to a small 14 kDa protein. No sample contained all 14 protein bands. Seven protein bands (101 kDa, 32 kDa, 26 kDa, 22 kDa, 18 kDa, 16 kDa, 14 kDa) were present in all samples, however the relative concentrations of protein within those bands varied between stallions. We demonstrated that although there is a characteristic equine seminal plasma protein profile on SDS-PAGE gels, there is between stallion variability in the relative amounts of each protein.     

SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the protein profiles of seminal plasma in buffalo bulls and to examine their correlation with semen characteristics. Semen of 10 buffalo bulls were collected by a bovine artificial vagina. Semen characteristics (motility, morphology, viability and concentration) were recorded. A part of the semen sample (1 ml) was diluted by tris-egg yolk-glycerol extender, packed in French straws and was frozen in liquid nitrogen. The straws were later thawed and semen characteristics were compared with those of the fresh semen. Seminal plasma was harvested by centrifugation; treated with cold ethanol and then, underwent SDS-polyacrylamide gel electrophoresis (PAGE). Twenty five protein bands were identified on the gel, of which those of <35.5 kDa were prominent (72% of the bands). Of these protein fractions, 24.5 kDa was significantly correlated with sperm progressive motility in fresh and viability in frozen-thawed semen while 45 kDa bands were correlated with abnormal morphology in frozen-thawed semen; 55 kDa protein fractions were correlated with sperm viability of fresh semen. Progressive motility, viability and abnormal sperm morphology of frozen-thawed semen were highly correlated with these parameters in the fresh semen. In conclusion, seminal plasma protein fractions in buffalo bulls are similar to those reported in other animal species and have some correlations with semen characteristics before and after freezing.     

Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the low weight (10-30 kDa) protein profile of bovine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine if any of these proteins was associated with semen freezability. Seminal plasma was collected from 16 bulls of high or low semen freezability. Twelve protein spots were identified from the 2D gel (15%); six of these were present in all samples. Of the 12 proteins found, three spots, present in all samples, 3 (15-16 kDa), 5 (16-17 kDa), and 7 (10-12 kDa) had nonsignificant variation among bulls, regardless of their freezability classification. Four proteins were more abundant (P<0.05) in seminal plasma samples collected from bulls with high semen freezability than in samples of bulls with low semen freezability: the spots 3 (15-16 kDa, pI 4.7-5.2), 7 (11-12 kDa, pI 4.8-4.9), 11 (13-14 kDa, pI 4.0-4.5), and 23 (20-22 kDa, pI 4.8-5.2). On the other hand, spot 25 (25-26 kDa, pI 6.0-6.5) was more abundant (P<0.05) on seminal plasma samples from bulls with low semen freezability. The N-terminus sequence of protein 7 was identical to the acidic seminal fluid protein (aSFP). Protein 23 (after trypsin digestion) had structural similarity to bovine clusterin. We concluded that there were differences in the seminal plasma protein profile from bulls with low and high semen freezability; aSFP, clusterin, proteins 3 and 11 may be used as semen freezability markers; and protein 25 was related to low semen freezability.     

大熊猫精液和包皮菌群组成及潜在致病菌调查

[目的] 大熊猫是我国国家一级保护动物,其种群面临着传染病和栖息地破碎化等持续威胁,其中生殖系统的细菌感染和菌群失衡会影响大熊猫生殖健康,严重者可导致流产,是引起大熊猫繁殖障碍的原因之一。本研究对精液与包皮分泌物样本的菌群组成情况及分离培养潜在致病菌开展研究。[方法] 通过采集13份大熊猫包皮分泌物和12份精液样本,采用16S rRNA扩增子测序技术、细菌培养及PCR鉴定的方法,确定样本中的细菌种类。[结果] 菌群组成分析结果显示,在门水平上,厚壁菌门（Firmicutes）的细菌丰度在大熊猫包皮与精液中均为最高;在属水平上,不同时期的雄性大熊猫包皮的菌群可能会发生改变,棒状杆菌属（Corynebacterium）和Dolosicoccus是Ⅰ期包皮样本中最丰富的微生物菌群,相对丰度分别为15.45%和12.40%;链球菌属（Streptococcus）和埃希氏菌属（Escherichia）是Ⅱ期包皮样本中最丰富的微生物菌群,相对分度分别为37.94%和9.68%;拟杆菌属（Bacteroides）和普雷沃氏菌属（Prevotella）是精液样本中最丰富的微生物菌群,相对丰度分别为14.40%和12.88%。菌群多样性分析结果显示,精液样品高于Ⅰ期包皮样品和Ⅱ期包皮样品,Ⅰ期包皮样品和Ⅱ期包皮样品之间无显著差异。通过细菌分离培养得到肺炎克雷伯菌（Klebsiella pneumonia）在内的多种潜在性致病菌。[结论] 本研究分析了大熊猫精液和不同时期包皮分泌物的菌群组成,其优势菌属存在差异,大熊猫包皮与精液中存在潜在性致病菌,这可能对大熊猫的生殖系统健康带来威胁,其致病性有待进一步研究。     

Vasectomy effect on canine seminal plasma biochemical components and their correlation with seminal parameters

Three semen samples were collected at 48 h intervals from 20 mature research dogs previously conditioned to manual semen collection. Vasectomy was performed in all dogs, and 15 days after surgery, another three ejaculates were similarly collected. The semen was evaluated, and centrifuged to obtain seminal plasma for measurement of pH, and concentrations of total proteins (TP), total chlorides (Cl), calcium (Ca), potassium (K), and sodium (Na). The seminal plasma protein profile was evaluated by SDS-PAGE; molecular weights and the integrated optical density (IOD) of each band were estimated. There was a negative correlation between K concentration and progressive motility (r = -0.49, P = 0.027), sperm vigor (r = -0.60, P = 0.0053), and plasma integrity, evaluated by both the hypo-osmotic swelling test (r = -0.50, P = 0.026) and a fluorescent stain (r = -0.45, P = 0.046). Positive correlations between Na and K pre- and post-vasectomy (r = 0.88, P < 0.001; r = 0.56, P < 0.01, respectively) were verified. There were a total of 37 bands pre-vasectomy and 35 post-vasectomy (range, 100.6-3.6 kDa). Bands B9 and B13 (42.6 and 29.2 kDa) were not present post-vasectomy. The IOD of band B3 (73.5 kDa) was higher (P = 0.03) pre-vasectomy, compared to post-vasectomy; conversely, the IODs of bands B29 and B37 (7.8 and 3.6 kDa) increased (P = 0.026 and 0.047). Pre-vasectomy, there was a positive correlation (r = 0.49, P = 0.029) between band B37 band (3.6 kDa) and the Na:K ratio. In conclusion, K appeared to be involved in sperm motility in dogs and could be a tool to evaluate sperm function. The prostate contributed several elements to canine seminal plasma. Vasectomy changed Ca concentrations and the protein profile of the seminal plasma. Further studies must be performed to clarify the function of these elements on the in vivo fertility of dogs.     

Ejaculates from the common marmoset (Callithrix jacchus) contain semenogelin and beta-microseminoprotein but not prostate-specific antigen

Human seminal plasma contains high concentrations of prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), beta-microseminoprotein (MSP), semenogelin I (SgI), and semenogelin II (SgII), whereas only PAP and MSP are present in rodents. In order to gain a better understanding of the evolution and function of semen proteins, we have studied ejaculates from the common marmoset (Callithrix jacchus)-a New World monkey. Semen samples were analyzed with SDS-PAGE, Western blotting, and isoelectric focusing. Under reducing conditions the dominating protein components appear as heterogeneous material of 55-70 kDa and distinct protein bands of 85, 17, 16, and 15 kDa. The heterogeneous material contains glycosylated material detected by an antiserum recognizing both human SgI and SgII. Southern blotting indicates that the common marmoset has genes for both SgI and SgII. There are several marmoset MSP genes, but the strong immunoreactivity against one 15 kDa semen component with pI 7.3 suggests preferential expression of one gene in the prostate. Expression of two other genes cannot be excluded as indicated by weak reaction to isoforms with pI 6.6 and 4.9. Unexpectedly, PSA was not detected by either immunological methods or activity measurements. This is in agreement with results from Southern blotting suggesting that the common marmoset might not have a PSA gene. Thus, in this study we have shown that semen coagulum proteins are present in marmoset seminal plasma, but the lack of PSA precludes a similar liquefaction as of human semen.     

大熊猫乳汁蛋白组成

测定了2只大熊猫（ailuropoda melanoleuca)乳汁中蛋白含量,乳糖含量,乳蛋白组成,并与成都麻羊（Capra hircus)初乳和中国荷斯坦牛（Bos taurus)乳进行了比较,大熊猫乳蛋白含量平均为41．52g/L,与中国荷斯坦牛乳接近;乳糖平均含量为15．41g/L,极显著低于牛乳和成都麻羊初乳,乳蛋白SDS－PAGE分析表明,大熊猫乳中酪蛋白（CN）含量明显低于羊乳和牛乳;乳中存在3条蛋白含量的区带,而在成都麻羊和中国荷斯坦牛乳电泳图谱的相应位置未见明显的区带,乳中上皮粘蛋白MUC1仅存在1条分子量约为196kDa的区带,未发现多态现象。     

Herapin-binding proteins of canine seminal plasma

Heparin-binding proteins (HBP) from seminal plasma have been expected to participate in modulation of the acrosomal reaction, and have been correlated with fertility in some species. However, they have not been described in the dog. The aim of this study was to document the HBPs of canine seminal plasma. Six pooled samples of seminal plasma from three crossbred dogs were used. The HBPs were isolated by heparin affinity chromatography and the fractions recovered were pooled. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 12 and 18% vertical minigels. The stained gels were scanned and the molecular weight (kDa) values for each band within a lane were calculated by image analysis software. The electrophoresis analysis of the pooled eluded fractions identified 19 bands, with molecular weights varying from 61.5 to 5.2 kDa. Previous studies, using one-dimensional SDS-PAGE, identified two bands (67 and 58.6 kDa), which were positively correlated with some semen parameters (sperm motility, sperm vigor, percentage of morphologically normal sperm and plasma membrane integrity). The 61.5 kDa band detected in the present study apparently corresponded to the 58.6 kDa band identified previously. Canine seminal plasma contained HBP; since HBP modulate the acrosome reaction in other species, they may have the same function in the dog. Further studies are necessary to better characterize this protein and determine if it is associated with fertility in the dog.