The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.     

Biometrics,Testosterone, Cortisol and Antler Growth Cycle in Iberian Red Deer Stags (Cervus elaphus hispanicus)

In this article, we aimed to describe the changes related to mating season in red deer, especially those related to antler growth, body condition score, testosterone and cortisol. Antler growth was studied in 17 Iberian red deer males, including body weight, antler length, biometric measures and testosterone and cortisol determination during 15 months. Body weight, body condition score, thoracic perimeter (TP), neck perimeter (NL) and testicular diameter (TD) showed the highest values immediately before mating season (autumn), decreased during it and remained constant at winter. Antler growth lasted 158 days and produced antlers with a final length of 80.8 ± 2.0 cm. Testosterone and cortisol showed seasonal changes with maximum values at September and May, respectively. Final antler size was related positively to cranial longitude, TP, NL, TD and body weight at casting time. No relationship between weight loss during precedent mating season and current antler size was found, but spring recovery weight was positively related to final antler size. Final length was related to the descent in testosterone values during previous mating season and to body weight before it. Spring recovery weight was related to relative weight loss during previous mating season. These results suggest that there is no relationship between the reproductive effort performed during one season and the next year size of the antler. In contrast, antler size was positively related to spring recovery weight, in the sense that those deer that recover a higher percentage of body weight at the early stages of antler growth develop higher antlers.     

Refrigerated Storage of Red Deer Epididymal Spermatozoa in the Epididymis, Diluted and with Vitamin C Supplementation

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 × 10⁶ sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 m m vitamin C. The remaining epididymides and the diluted samples were stored at 5°C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.     

Paratuberculosis in Red Deer (Cervus elaphus hippelaphus) in the Western Alps

During the hunting seasons 1995–96 to 1997–98, 19 red deer from the Upper Susa Valley (Cottian Alps) were examined for paratuberculosis (Johne's disease). Specific DNA amplification on mesenteric lymph nodes detected Mycobacterium avium paratuberculosis in 17 animals. Ten of these red deer were tested for serum antibodies by the AGID and ELISA tests, nine being negative. Three isolates from infected deer were genetically characterized by an arbitrarily primed polymerase chain reaction, and showed similar genetic polymorphism to that of bovine strains isolated in different Italian areas. The study showed that paratuberculosis is present in red deer of the Upper Susa Valley and that serological tests are not an efficient means for monitoring this infection.     

中国马鹿的研究现状及展望

马鹿在世界上主要分布于欧洲南部和中部、北美洲、非洲北部、亚洲的俄罗斯东部、蒙古、朝鲜和喜马拉雅山地区,在中国马鹿有8个亚种,主要分布在东北、华北、西北及西南等地。本文根据文献资料系统地总结了中国马鹿的研究现状,展望了马鹿需要研究的问题,为中国马鹿的保护及科研活动开展提供依据。     

马鹿的起源进化与遗传多样性研究进展

马鹿是非常重要的物种资源,由于栖息地的流失和人为干扰进行近亲繁殖等导致野生马鹿数量急剧减少,而家养马鹿多经过改良,因此马鹿的纯种数量锐减。对马鹿进行分子遗传学研究不仅可以加深人们对马鹿起源和物种形成的认识,还能帮助开展遗传多样性保护研究。随着高通量测序技术、分子生物学和生物信息学的迅速发展,马鹿的起源进化研究已发展到全基因组水平,并取得了一定的成果。马鹿的起源进化研究从最初对体态外貌和染色体核型的研究逐渐发展到对DNA序列与生理指标的研究。文章回顾了近年来国内外对马鹿起源进化和遗传多样性方面的研究,从起源时间、起源地和迁徙路线等方面揭示了马鹿的演化历史,介绍了父系、母系和常染色体研究方面分析了马鹿遗传多样性选取的不同分子标记,为进一步揭示马鹿种群的遗传变异、分化情况、迁徙路线和系统发育关系等提供基础信息,同时为马鹿遗传资源的利用和保护以及马鹿产业的良性发展提供重要的借鉴与参考。     

Histological Studies of Growing and Mature Antlers of Red Deer Stags (Cervus elaphus)

This study aims at presenting histology of growing and mature antlers in red deer stag ( Cervus elaphus ). Growing antlers constitute a model organ for examining regeneration processes of tissues because they are the only mammalian appendages capable of regeneration. Histological study revealed that the tip of a growing antler consists of hairy skin, perichondrium, mesenchyme and chondroprogenitors area. By performing immunochistochemistry, we found that cell expressing Ki-67 and PCNA antigens were localized in basal layer of epidermis, skin glands and beneath their secretory sections, mesenchyme as well as within and in the vicinity of central blood vessels. Ultrastructurally, cells from chondroprogenitors zone have chondroblast-like morphology and take part in producing of collagen fibres followed by the process of cartilage mineralization. However, mature antlers also consist of lamellar osseous tissue.     

基于线粒体Cyt b DNA阿拉善马鹿分子系统学研究

阿拉善马鹿为国家Ⅱ级重点保护野生动物,仅分布于宁夏和内蒙古交界的贺兰山中段,是我国唯一幸存的该亚种的有效种群。目前关于野生阿拉善马鹿的分子研究尚属空白,本研究于2016年8—9月和2017年2—3月在贺兰山采集新鲜阿拉善马鹿粪便样本396份,使用线粒体Cyt b区DNA引物对提取的DNA进行物种鉴定和9对微卫星引物对其个体识别,共鉴定出278个阿拉善马鹿个体,基于线粒体Cyt b DNA对其全序列进行分析。使用线粒体Cyt b区引物对提取的粪便DNA扩增,获得了1 075 bp的序列,检测到10个变异位点,并定义了10个单倍型。其中10个变异位点占分析长度的0. 93%,均为碱基代换,无碱基插入和缺失。其单倍型多样性为0. 243,核苷酸多样性为0. 000 32。中性检验Tajima's D值为显著负值,Fu和Li's D值为不显著的负值,表示阿拉善马鹿可能经历瓶颈事件并随后出现种群扩张。在系统发育树中,阿拉善马鹿单独聚成一支且与东北马鹿亲缘关系较近,与其他中国马鹿亚种亲缘关系较远。本研究表明野生阿拉善马鹿种群总体遗传多样性较低,建议加强对该亚种的保护力度。     

Effects of Egg Yolk Source on the Cryopreservation of Stallion Spermatozoa

Three experiments were conducted to determine whether replacement of chicken egg yolk, as a component of freezing extenders, with egg yolk from other avian species would improve the post-thaw motility and percentage of intact acrosomes of stallion spermatozoa. In the first experiment, substitution of chicken egg yolk with chukar egg yolk, as a component of the lactose-ethylenediaminetetraacetic acid extender, improved (P ≤ .05) the post-thaw motility of stallion spermatozoa. These results were not replicated in (IMV Technologies, Maple Grove, MN, USA) a more expansive study comparing 2%, 4%, 6%, or 8% egg yolk combined with INRA 96 when a “slow freeze” method was used, or the same substitution at levels ranging from 13% to 22% when egg yolk was combined with lactose-ethylenediaminetetraacetic acid for diluents used for a “fast freeze” method of cryopreservation. In the third study, egg yolks from regular and high omega-3 chicken eggs as well as from turkey, chukar, and mallard duck eggs were analyzed for lipid content and fatty acid profile. The yolk from the turkey eggs was higher (1,300 mg/100 g) and that from mallard ducks was lower (560 mg/100 g) in cholesterol as compared with the two types of chicken eggs and chukar egg yolk (range, 1,046-1,094 mg/100 g). In addition, the high omega-3 eggs did test higher for fatty acids (4.51 g/100 g) than other types of eggs (range, 0.28-0.73 g/100 g). Substitution of chicken egg yolk with turkey, but not duck, egg yolk resulted in higher post-thaw total motility (P ≤ .05) for spermatozoa obtained from two of the three stallions used in the third experiment.     

阿拉善马鹿冬季肠道菌群组成的性别差异

肠道菌群对于动物消化及健康有着至关重要的作用,但在性别差异上的研究仍然有限。本研究通过高通量测序技术对贺兰山阿拉善马鹿(Cervus elaphus alashanicus)的20个粪便样本进行多样性分析,雌雄各半,并讨论性别因素对肠道菌群的影响。结果表明,阿拉善马鹿雌雄肠道菌群的多样性及结构均存在差异,且雌性肠道菌群的多样性更高。菌群构成的门水平上,雄性肠道菌群的优势菌门为厚壁菌门(Firmicutes 49.47%)和疣微菌门(Verrucomicrobia 14.21%),雌性肠道优势菌门为厚壁菌门(54.47%)和变形菌门(Proteobacteria 54.41%)。在属水平上,雌雄肠道菌群的优势菌属都为孢杆菌属(Sporobacter),含量为(雌性:6.79%,雄性:7.97%),拟杆菌属(Bacteroides),含量为(雌性:3.87%,雄性:4.42%),NMDS分析及LEfSe分析表明,性别因素对阿拉善马鹿雌雄肠道菌群的影响存在显著差异(P<0.05)。本研究表明性别因素对阿拉善马鹿冬季肠道菌群的多样性及构成存在影响,可为深入研究性别因素对阿拉善马鹿肠道菌群的影响提供依据。     

基于RNA-Seq技术的塔里木马鹿毛色相关差异表达基因筛选及初步分析

旨在基于RNA-Seq技术对塔里木马鹿毛色相关基因进行筛选及分析。采用Illumina Hi Seq TM2000测序平台对塔里木马鹿和天山马鹿的皮肤组织进行转录组测序,所得序列经质控、组装后比对到NR、Swiss-Prot、COG、KOG、KEGG、GO和Pfam数据库中注释,并对差异表达基因进行筛选、功能注释和富集分析。结果表明,测序获得25 038个有注释信息的Unigenes,比对分析显示,塔里木马鹿与天山马鹿有922个差异表达基因,其中上调表达基因495个,下调表达基因427个;GO功能富集分析结果显示,568个差异表达基因富集到61个GO条目上,分别参与了生物学过程、细胞组分及分子功能;KEGG代谢通路富集分析发现,在差异表达基因中富集最显著的代谢通路是ECM-受体相互作用。利用实时荧光定量PCR（qRT-PCR）方法分析与塔里木马鹿毛色相关的7个候选基因的转录水平变化来验证转录组测序结果的准确性和可靠性,这些基因的表达趋势与转录组测序结果相一致。ECM-受体相互作用、蛋白质消化与吸收、PI3K-Akt信号通路及与黑色素合成相关的酪氨酸等通路可能与塔里木马鹿的毛色有关;候选基因MITF、Ggt1、VDR、PTPRF、CⅡTA、ARPC5L、POMC等可能在塔里木马鹿毛色形成过程中发挥重要作用。本研究结果为今后塔里木马鹿毛色相关基因的分子调控机制方面及挖掘潜在的新基因提供了丰富的试验数据。     

鸵鸟卵黄低密度脂蛋白对猪精子冻后品质的影响

在家畜精液冷冻中,卵黄被广泛应用,且其中的低密度脂蛋白(LDL)对精子起主要保护作用。本研究利用含6%、7%、8%和9%鸵鸟卵黄LDL配制的稀释液制作猪细管冷冻精液,分析鸵鸟卵黄LDL对冷冻-解冻后猪精子质量参数的影响。结果表明:在含不同浓度鸵鸟卵黄LDL的稀释液中,8%LDL的稀释液冷冻效果最好,冻后精子活率平均可达52.13%,显著高于其他组(P<0.05);精子顶体完整率平均为58.33%,质膜完整率为72.38%,与其他处理组相比差异显著(P<0.05)。但与鸡蛋卵黄LDL和鸽子蛋卵黄LDL处理组相比,鸵鸟卵黄LDL处理组冷冻-解冻后猪精子质量参数相对较低。本研究表明,虽然鸵鸟卵黄LDL在冷冻过程中对猪精子具有一定的保护作用,但相对于鸽子蛋和鸡蛋卵黄LDL效果并不理想。     

基于粪便分子生物学技术的中国马鹿分子生态学研究进展

中国马鹿分为东北亚种、天山亚种、塔里木亚种、甘肃亚种、川西亚种、阿拉善亚种、西藏亚种和阿尔泰亚种。根据文献资料系统性总结了以粪便为研究材料对中国马鹿各个亚种的分子生态学的研究现状,展望了中国马鹿分子生态学需要研究的问题;具体介绍了马鹿分子生态学中研究很少的亲权鉴定、近亲交配和家域利用方面的研究现状并提出了进一步的研究趋势。为中国马鹿的种群恢复、保护管理以及科学研究提供依据。     

岩羊和马鹿对人与机动车反应的差异分析

人为干扰会对野生动物种间关系、个体适合度、群落结构和繁殖成功率等产生中长期的影响。因此,研究野生动物反干扰行为对于我们认识该物种对其生境的行为适应和进化机制具有重要意义。2017年11—12月在内蒙古贺兰山国家级自然保护区内通过建立多元逻辑斯蒂回归模型研究了岩羊(Pseudois nayaur)、马鹿(Cervus elaphus)的反干扰策略,本研究共设置33条样线,记录了10种在人为干扰下可能会影响岩羊、马鹿反应行为的变量,经模型分析发现影响岩羊反应行为的变量有5种,影响马鹿反应行为的变量有4种,共同影响因子分别是干扰源、性别、头的朝向和地形特征,而植被类型则只对岩羊产生影响。最后根据逻辑模型得出的数据计算发生比,从而了解各个分类变量与反应行为的关系。     

Milk production and composition in captive Iberian red deer (Cervus elaphus hispanicus): effect of birth date

This study describes milk production and milk composition of Iberian red deer (Cervus elaphus hispanicus) females (hinds) and the effect of calving date and BW change of hinds by milking over 34 wk. All hinds produced milk throughout the 34-wk study period, well over the standard lactation period. Total milk yield was 224.1 +/- 21.1 L, and daily production was 0.91 +/- 0.06 L. Milk yield decreased with lactation stage (P = 0.01) and the later a calf was born (P = 0.008), and it was greater in posterior quarters (P < 0.05). Milk yield was unaffected (P > 0.10) by side position or milking order of the udder. Milk production did not correlate with hind BW (P > 0.1). Hinds lost 4.4% BW during lactation (P < 0.001); losses increased the later a calf was born (P = 0.012). Iberian red deer milk had 11.5% fat, 7.6% protein, 5.9% lactose, and 26.7% DM. Stage of lactation affected fat (P < 0.001), protein (P = 0.002), DM (P < 0.001), and protein:fat ratio (P < 0.001), but not lactose (P > 0.1). These constituents became concentrated as lactation proceeded, and protein was substituted by fat. Calving date had a similar concentrating effect on fat (P < 0.001) and DM (P < 0.001), whereas it decreased lactose (P = 0.015) and protein (P = 0.002), thus producing a substitution of protein by fat (ratio of protein to fat, P < 0.001). Milking order of quarters or their position had no effect on milk composition (P > 0.10). Results suggest that milk production and milk energetic quality might increase by advancing calving date in red deer hinds.     

Growth marks observation: preferential techniques and teeth for ageing of Spanish red deer (Cervus elaphus hispanicus)

A total of 207 teeth belonging to 53 deer (Cervus elaphus hispanicus) from Sierra Morena (South of Spain) were analysed using histological thin sections and/or polished thick sections. Growth marks were observed in dentine and cementum of incisors, molars and canines. Tooth, region of the tooth, and type of preparation giving the best performance in terms of sharpness and interpretation ease of the growth marks, when used for Spanish deer ageing, were identified. The tissue chosen for the study was the cellular cementum of the inter-radicular pad from molars and the acellular cementum of the root from incisors, both observed through longitudinal cuts. Histological sections obtained with a conventional microtome were optimal for a reliable identification of the first growth marks. In sections thicker than 15 microm the first rest line, when observed in cementum can be confused with the cementum-dentine junction; whereas in the polished sections from molars it will not appear at all. Dentine interpretation was more difficult. Its observation should only be accomplished using cross-sections of the molar root. Secondary or accessory marks were visible in every tooth, being those from the molar pad the easiest to distinguish. Canines were the less suitable teeth for this study.     

Histological thin-sections: a method for the microscopic study of teeth in Spanish red deer (Cervus elaphus hispanicus)

A method to obtain thin histological sections (4-5 microm thick) from teeth, using a conventional microtome, is described for incisors, molars and canines from Spanish red deer (Cervus elaphus hispanicus). Decalcifying solutions of formic acid and nitric acid at different concentrations were ineffective. This was not the case for a solution of 14% hypochloric acid and polyvinylpyrrolidone (TBD-1(R) of Shandon). However, the time needed to complete decalcification was longer than the time estimated by other authors. Incisors required between 4 and 6 days, molars between 7 and 15 days, and canines between 8 and 12 days. The preparation of histological thin-sections from teeth with a conventional microtome, required some training or prior specialization, because most of defective or lost material occurred during the preliminary stage of the study. However this technique is inexpensive enough to be implemented in any histological laboratory. The preparations obtained are suitable for growth mark observation in dentine and in cementum for dating studies. Nevertheless, they are not suitable for enamel change studies, because the enamel is completely destroyed during the decalcifying process.     

Heterologous In Vitro Fertility Evaluation of Cryopreserved Iberian Red Deer Epididymal Spermatozoa with Zona-intact Sheep Oocytes and its Relationship with the Characteristics of Thawed Spermatozoa

A heterologous in vitro system, using zona‐intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37°C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (～80, 80 and 70% respectively). However, these values significantly decreased after incubation (～59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona‐intact sheep oocytes, although the percentage of cleaved oocytes was low (～22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona‐intact sheep oocytes.     

STR在野生东北马鹿个体识别中的应用

为了建立有效的东北马鹿个体识别体系,从12对加拿大马鹿的微卫星引物和6对黑尾鹿的微卫星引物中,筛选出8个具有多态性的微卫星位点。各位点的观察杂合度为0．3314～0．8174,平均杂合度为0．6243;各位点的匹配率为0．0656～0．5130,累计匹配率为1．446×10^-6;各位点的识别率为0．4870～0．9344,累计个体识别能力为0．9999。这8个微卫星位点可以准确地进行东北马鹿的个体识别分析。