Multivariate Cluster Analysis Regression Procedures as Tools to Identify Motile Sperm Subpopulations in Rabbit Semen and to Predict Semen Fertility and Litter Size

Computerized motility analysis (CASA) shows that four separate subpopulations of spermatozoa with different motility characteristics co-exist in rabbit ejaculates. There were significant (p < 0.01) differences in the distribution of these subpopulations among separate genetic lines, total sperm abnormalities and the percentage of altered acrosomes. Furthermore, logistic and linear multivariate regressions among several parameters of rabbit semen quality analysis were tested for use as predictive tools for the fertilizing ability of a specific artificial insemination semen sample. Logistic regression analysis rendered two mathematical, significant (p < 0.01) models: one between sperm viability and conception rate and the other between total sperm abnormalities and conception rate. Multiple linear regression analyses also yielded some significant relationships between both fertility (p < 0.001) and litter size (p < 0.05), with respect to some semen characteristics. Our results support the hypothesis that the predictive in vivo fertility use of the standard rabbit semen quality analysis coupled with a CASA determination could be reasonably achieved by applying linear and logistic regression analyses among several parameters of rabbit semen quality analysis.     

品种、季节、胎次、采精月龄及采精间隔对猪精液品质的影响

【目的】探究品种、公猪出生胎次、公猪同窝仔猪数、公猪乳头数、采精季节、采精月龄和采精间隔对猪精液品质的影响,以及不同品种对精液质量稳定性的影响。【方法】选取909头杜洛克、长白、大白种公猪为试验群体,收集2021年4月至2022年4月27 408条精液测定记录,采用混合线性分析模型和方差分析探究品种、公猪出生胎次、公猪同窝仔猪数、公猪乳头数、采精季节、采精月龄和采精间隔对精液体积、精液密度、精子活力、直线前进运动精子比例、精子畸形率、总精子数及各精液性状稳定性的影响。【结果】从不同品种对精液品质的影响来看,长白猪精液体积和总精子数均显著高于大白猪、杜洛克猪(P<0.05),杜洛克猪精液密度显著高于大白猪和长白猪(P<0.05),杜洛克猪、大白猪精子活力均显著高于长白猪(P<0.05),大白猪直线前进运动精子比例显著高于长白猪和杜洛克猪(P<0.05),长白猪和大白猪精子畸形率均显著低于杜洛克猪(P<0.05);从不同公猪出生胎次对精液品质的影响来看,1～3胎出生的公猪具有较高的精液品质;从不同采精季节对精液品质的影响来看,精液密度、精子活力和总精子数秋、冬...     

Use of a sperm quality analyser on semen of turkey breeders to monitor storage time effects and age-related changes during a reproductive cycle

1. A relatively new instrument known as a Sperm Quality Analyzer (SQA) offers a rapid assessment of sperm quality and quantity by providing a sperm quality index (SQI). The SQA measures the intensity of sperm activity and motile concentration by determining the number and amplitude of sperm movements per second in a capillary tube as detected through light beam interference. 2. The objectives of the current study were to determine if the SQA could accurately reflect changes in semen quality that occur with prolonged storage of semen and to determine the variation and change in SQI values among individual breeding male turkeys during their semen production cycle. 3. The effect of storage time on SQI values was evaluated by diluting semen with extender and placing the semen on an oscillating shaker at 4 degrees C for 8 h. The SQI values and sperm viability, expressed as % dead sperm, were recorded hourly. The SQI readings declined linearly with increased storage time while % dead sperm increased linearly with increased semen storage. 4. Semen from 220 individual males was analysed monthly for 9 months. Semen diluted 50-fold with saline had lower SQI values during pre- and post-peak phases of production (months 1, 7, 8, and 9 as compared with months 2 to 6 of semen production). The highest SQI values occurred during months 2 to 6. The largest variation in SQI values occurred during months 1 (CV = 26%) and 9 (CV = 31%) with a CV that averaged 16% for the remaining months. 5. Correlation analysis of SQI values for each bird averaged over 9 months with individual male SQIs for each month showed monthly correlation coefficients that ranged from 0.22 to 0.63. 6. These results indicate that the SQA accurately assessed the decline in sperm quality that occurs with prolonged storage of turkey semen and reflected age-related changes in semen quality and quantity that occurred during a semen production cycle of turkey breeders. In addition, the semen quality rank of some turkey breeders in a population changed with age.     

Rotation of Boar Semen Doses During Storage Affects Sperm Quality

It is common practice to rotate boar semen doses during storage for prevention of sperm sedimentation. In this study, the effect of rotation of boar semen doses during storage on sperm quality was investigated. Manual turning twice daily and automatic rotation five times per hour resulted in the following effects: alkalinization of the BTS‐extender, loss of membrane integrity at day 3, and loss of motility and changes in sperm kinematics during a thermoresistance test at day 5. Using a pH‐stabilized variant of BTS extender, sperm motility and velocity decreased in continuously rotated samples, whereas membrane integrity and mitochondrial activity remain unaffected. It is concluded that rotation of semen samples adversely affects sperm quality and, therefore, should no longer be recommended for AI practice.     

Changes in Semen Quality in Estonian Holstein AI Bulls at 3, 5 and 7 years of Age

The predictability of semen quality of mature sires from measurements at an early age is not well established. The aim of the present study was to determine age-dependent changes in the quality of bull semen from six Estonian Holstein (EHF) bulls, processed when the sires were 3, 5 and 7 years old. Fertility data such as 60-day non-return to oestrus rates (60d-NRRs) were available for 3-year-old bulls. From each batch, semen straws were analysed immediately after thawing [i.e. post-thaw (PT)] (controls) and after a swim-up (SU) procedure. The analyses comprised subjective and computerized measurements of sperm motility using computer-assisted sperm analysis (CASA) as well as estimations of sperm concentration, morphology and membrane integrity. There was a significant (p < 0.05) increase in the percentage of sperm motility (SU), membrane integrity (PT, SU) and normal tail and acrosome morphology (SU) with an increase in the age of the sires. The percentage of total motile spermatozoa PT measured by CASA correlated between 3- and 7-, and between 5- and 7-year-old bulls (p < 0.05). In addition, the proportion of head abnormalities tended to correlate between all three age groups both PT and after SU (p < 0.1). The sperm parameters correlating with fertility were average path velocity (VAP) (p < 0.001), total motility as measured by CASA (p < 0.01), linearly motile spermatozoa (p < 0.05) and CASA-assessed numbers of motile spermatozoa (p < 0.05), all after SU selection. The results showed that overall semen quality examined at 3 years of age is related to the semen parameters later in bulls' life. Moreover, CASA-assessed motility after SU seems to be a reliable marker for semen quality assessment as it shows correlation not only between the ages, but also to field fertility.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

不同品种、月份、月龄及采精间隔对猪精液品质的影响

为研究不同品种、采精月份、采精月龄和采精间隔等因素对加系公猪精液品质的影响,以及品种、初次采精周龄对精液质量稳定性的影响,本研究以江西某种公猪站79头加系大白猪、长白猪、杜洛克猪种公猪为试验群体,收集2018年12月至2020年12月3 921条精液采集与精液质量数据,通过混合线性模型与方差分析探究各因素对精液量、精液密度、精子活力、总精子数及其稳定性的影响。结果显示,从不同品种对精液质量的影响来看,长白猪精液量和总精子数均高于大白猪、杜洛克猪,但杜洛克猪精液密度高于长白猪、大白猪,杜洛克猪精子活力最低;从不同月份来看,1~3月采精精液密度最高,4~6月采精精子活力最高,10~12月采精精液量和总精子数最高,精液量呈现秋冬多、春夏少的季节变化规律。公猪不同月龄采精,精液质量指标也存在差异,月龄越小精液量越低,但精液密度偏高,精子活力相对较好,在19~24月龄黄金期总精子数最高。不同采精间隔对精液质量有较大影响,采精间隔越长,精液量、精液密度、精子活力和总精子数相对较好,采精间隔为5 d时综合性能最佳,但过长的采精间隔导致精子活力降低。品种影响总精子数稳定性,长白猪、大白猪总精子数稳定性显著优于杜洛克猪（P<0.05）。本研究结果表明,品种、采精月份、采精月龄和采精间隔均会影响公猪精液质量,关注这些因素有助于公猪站制定更完善的生产计划,提高公猪利用率。     

气候因素对公犬采精量及精液品质的影响

随机选取南方某犬场使用的德国牧羊犬种公犬16头,对该批种公犬在2011年度所采集的精液量及精液品质进行统计,发现当地的气候(气温、降雨量)变化对公犬采精量及精液品质有直接影响。结果表明,公犬精液品质会随着采精月份及季节的不同而发生一定变化,夏季高温环境对公犬精液品质影响较大,精液品质下降;精子活力、精子密度及精子畸形率与气温的变化呈中等相关(P<0.1),射精量与气温变化的相关不明显(P>0.1),而射精量与降雨量的变化呈中等相关(P<0.1),与其它性状相关不明显(P>0.1)。     

Phenotypic Correlations of Testes Size with Semen Traits and the Productive Traits of Young Boars

The aim of this study was to investigate whether there is a relationship of young boar testes size with semen traits and with productive traits. The dimensions (length, width and volume) of each testis and semen traits (semen volume, percentage of sperm with progressive motility, sperm concentration, total number of sperm in semen, percentage of sperm with normal acrosome, percentage of sperm with major and minor morphological defects, osmotic resistance test value and activity of aspartate aminotransferase in seminal plasma) were determined on 120 young boars aged 6 months. At 180 day of age, the boars backfat thickness and leanness (by ultrasonic apparatus) and body weight were also measured. The average daily gain was determined in the period from 70 to 180 days of age of the boars. The results showed that the sperm concentration, total number of sperm in semen and percentage of progressive motile sperm were a significantly positively correlated with width and volume of the left (p ≤ 0.01) and right testis (p ≤ 0.05) and with total volume of both testes (p ≤ 0.01). But the highest values of correlation coefficients were found between the width of the left testis and sperm concentration, total number of sperm in semen and percentage of progressive motile sperm. A correlations of dimensions (length and width) and volume of testes with other semen traits were very low and statistically non‐significant. The volume of testes (left and right testis and total testes) was significantly positively correlated with body weight at 180 days of age and daily gain (p ≤ 0.01), but lower correlation coefficient was between left testis and daily gain (p ≤ 0.05), whereas correlations were low and non‐significant with leanness and backfat thickness.     

Influence of the Preservation Temperature (37, 20, 4, −196°C) and the Mixing of Semen over Sperm Quality of Majorera Bucks

This study assessed the effect of different semen storage temperatures and the influence of semen pooling in semen viability. In experiment 1, semen samples (n = 30) of five Majorera bucks were individually processed [Individual semen (IS)] and after the first dilution (Tris‐yolk extender), semen‐diluted aliquots from each male were pooled semen (PS). Thereafter, semen samples (IS and PS) were preserved as fresh semen (37 and 20°C), chilled semen (4°C) and frozen semen. Sperm motility and the percentage of abnormal sperm cells and intact membrane acrosomes were defined. Semen preservation at 20 and 4°C did not modify the quality of spermatozoa for the first 24 h, but the conservation at 37°C caused a dramatic fall in the semen motility from 12 h onwards. Furthermore, the longevity of frozen‐thawed semen was limited to 4–6 h. No differences were observed in semen parameters when PS was compared with semen from individual males in any of the preservation protocols assessed. In experiment 2, 120 goats were distributed in four experimental groups: in group fresh individual semen (FIS, n = 30) and group frozen‐thawed individual semen (FTIS, n = 30), does were transcervically inseminated with fresh semen and frozen‐thawed semen from each individual male, respectively, and in group fresh pooled semen (FPS, n = 30) and group frozen‐thawed pooled semen (FTPS, n = 30), goats were transcervically inseminated with FPS and FTPS, respectively. The kidding rate was very close in the FIS and FPS groups (70.0% and 73.7%, respectively), and no significant differences were observed in the fertility rate between FTIS and FTPS. The results of this study confirmed that semen samples may be preserved satisfactorily for 24 h both at 20 and 4°C. In addition, the mixture of semen of different bucks did not significantly modify the semen parameters when compared with semen from individual males.     

Validation and Usefulness of the Sperm Quality Analyzer (SQA II-C) for Bull Semen Analysis

In this study, an upgrade version of the Sperm Quality Analyzer (SQA), the SQA-IIC was tested for the assessment of bull semen quality. In Expt 1, the device showed good repeatability of measurements within and between capillaries, as evidenced by the low coefficients of variation (CVs; < 13%) at concentrations between 35 and 705 x 10(6) spermatozoa/ml. In Expt 2, 10 semen concentrations (1-1000 x 10(6)/ml) were stored in HEPES TALP for 48 h at room temperature. A time-dependent decrease in sperm motility index (SMI) values was noticed. SMI values increased linearly with increasing sperm concentrations, but remained constant around 500, corresponding to a concentration of approximately 50 x 10(6)/ml. For sperm concentrations below 50 x 10(6)/ml, SMI values were highly correlated with concentration (p < 0.05) and with semen parameters, expressing the overall semen quality (p < 0.05; Expt 3). In Expt 4, a correlation of only 0.44 (p < 0.05) between SMI values of frozen-thawed semen samples of 35 bulls and the corrected 56-day non-return rate (56dNRRc) was found. Prediction of the 56dNRRc based on the SMI value of a semen sample was inaccurate. The present study indicates that the SQA-IIC is suitable for a rapid screening of bull semen diluted to a concentration of approximately 50 x 10(6)/ml. Furthermore, the device seems inappropriate for fertility prediction.     

Effects of Incubation Temperature and Semen Pooling on the Viability of Fresh,Chilled and Freeze‐Thawed Canine Semen Samples

This study assessed the effects of different incubation temperatures on semen viability and the influence of pooling on semen longevity. In experiment 1, semen samples were collected from five dogs, individually processed (individual semen: IS) and then aliquots from each male were pooled (pooled semen: PS). Semen samples (IS and PS) were diluted in a Tris‐glucose‐yolk extender and preserved as fresh (37 and 25°C) and chilled semen (4°C). Sperm motility and the percentages of sperm abnormalities and acrosome membrane integrity were assessed for 24 h. Storage at 25 or 4°C for the first 24 h yielded similar semen quality, but incubation at 37°C caused drastic reduction in sperm motility from 8 h of incubation onwards. In experiment 2, the semen was processed in the same way to that of experiment 1 and then preserved at 25 or 4°C until semen inactivation. Semen that was incubated at 25°C became completely inactive after 3–4 days of storage, while semen that was preserved at 4°C presented with more gradually decreased sperm motility (mean values of 40–60% for the first 8 days). In addition, the mixing of semen was only observed to influence the sperm quality of the samples stored at 4°C. In experiment 3, semen was collected from five dogs, pooled and frozen in liquid nitrogen; after thawing, it was preserved at 37, 25, 15 and 4°C, and the sperm quality was defined. The motility of the freeze‐thawed semen samples decreased quickly in the first 4 h after thawing, regardless of the preservation temperature of the thawed semen. This study confirmed that semen preserved at 37°C should be used within a maximum of 12 h, while the semen stored at 25°C shows acceptable quality for 24 h. Chilled semen presented highest most sustainable quality, especially when semen is processed as pooled semen.     

Fertilizing Ability of Electro-Ejaculated Cryopreserved Semen in the Domestic Cat

Semen collection and AI in the cat are still not routine procedures. The correlation between semen quality and fertility under natural conditions is a relatively unknown field in the cat. In the present study, functional in vitro tests, such as the ability to bind and penetrate the zona pellucida or to fertilize in vitro, were used to determine fertilizing ability of sperm cryopreserved with a practical and efficient freezing protocol previously developed in our laboratory. Semen was collected by electroejaculation, evaluated for motility and diluted with Tris-glucose-citrate egg-yolk extender supplemented with Equex STM paste (0.5% v/v). After equilibration and loading into 0.25 ml straws, semen was frozen at 3.85 degrees C/min. Frozen-thawed semen was co-cultured with in vitro matured cat oocytes. Penetration rate was recorded 30 h after in vitro fertilization and cleaved zygotes were cultured in vitro until day 7. A correlation was found between sperm motility index (SMI) after thawing and semen fertilizing ability (p<0.05). In conclusion, it was demonstrated that the post-thaw motility quality, expressed as SMI, of spermatozoa frozen using the protocol mentioned above can be considered an index of the sperm ability to penetrate in vitro matured oocytes.     

Numbers of Sertoli cells, quantitative rates of sperm production, and the efficiency of spermatogenesis in relation to the daily sperm output and seminal quality of young beef bulls

Data from 34 yearling Hereford or Angus bulls were used to investigate relationships of testicular size, quantitative rates of sperm production, Sertoli cell numbers, numbers of germ cells supported per Sertoli cell, and the efficiency of spermatogenesis to daily sperm output and seminal quality. Two ejaculates were collected by electroejaculation from each bull on each of 2 days/week throughout the study. The percentage of progressively motile sperm and the percentage of morphologically normal sperm were determined from aliquots of fresh semen. Additional aliquots of semen were frozen in glass ampules or plastic straws and subsequently evaluated for postthaw motility and percentage of sperm with intact acrosomes. Sertoli cell numbers, the numbers of germ-cells per Sertoli cell, and the efficiency of spermatogenesis were unrelated to the quality of fresh or frozen semen (P greater than 0.05). In first ejaculates, the numbers of sperm and motile sperm were related (P less than 0.05) to testicular parenchymal weight (r = 0.38 and 0.50), daily sperm production (r = 0.45 and 0.53), and spermatids per gram of testicular parenchyma (r = 0.35 and 0.34). Testicular parenchymal weight and daily sperm production also were related to daily sperm output and to the average daily motile sperm output of these bulls (P less than 0.05), but could account for less than 25% of the variability in these end points among bulls.     

鸡精液稀释配方对低温保存精子活力和输精效果的影响

为了完善中国南方地区鸡场公鸡精液的保存技术、提高精液的利用率，本试验研究了在低温（4 ℃）保存的条件下，不同的保存时间（0、4、8、24 h）、不同的稀释液配方（原精液、配方Ⅰ和配方Ⅱ）对精液的精子活力变化以及人工输精繁殖效果的影响。选用33周龄黄鸡母鸡192只、公鸡42只，192只母鸡依笼号分为12组（3种处理精液×4个保存时间），每组16只母鸡，公鸡不分组。统一采精后用两种不同稀释液稀释、低温（4 ℃）保存至0、4、8、24 h后观察精子活力，并对母鸡输精，分组收集鸡蛋，对各组受精率、出雏率、健雏率进行比较分析，以原精液低温保存作为对照。结果显示，两种稀释液组的精子低温（4 ℃）保存4、8、24 h，其精子活力极显著高于原精液组（P<0.01），配方Ⅱ组精子的活力高于配方Ⅰ组（P>0.05）；两种配方稀释液组的精液低温（4 ℃）保存4 h，输精受精率高于原精液组（P<0.05）；两种配方稀释液组的精液在低温（4 ℃）保存8 h，输精受精率极显著高于原精液组（P<0.01）。表明这两种精液稀释液更有利于精液保存，经稀释后的精液可以显著提高受精率，可为中国南方鸡场种公鸡精液保存技术的完善提供有力的数据支撑。     

Multivariate Analyses for Determining the Association of Field Porcine Fertility With Sperm Motion Traits Analysed by Computer‐Assisted Semen Analysis and With Sperm Morphology

This study investigates the association of semen traits with boar fertility. The fertility outcome (farrowing rate – FR and total piglets born – TB) of 14 boars was obtained from a field trial conducted during 10 week of breeding period on a commercial farm using multiparous sows (n = 948) through single‐sire mating with 2 × 10⁹ motile sperm cells per artificial insemination (AI) dose. Sperm motion parameters, evaluated with computer‐assisted semen analysis system in raw and stored semen at 17°C for 240 h, in addition to morphological sperm defects, measured on the collection day, were included in the analysis to determine which semen traits were important to discriminate the fertility potential of ejaculates from these boars. The data underwent multivariate cluster, canonical and discriminant analyses. Four clusters of boars were formed based on fertility outcome. One boar, with the lowest FR and TB values (89.7% and 11.98), and two boars, with the highest FR and TB values (97.8% and 14.16), were placed in different clusters. The other boars were separated in two distinct clusters (four and seven boars), including boars with intermediate TB (12.64 and 13.22) but divergent values for FR (95.9% vs 91.8%). Semen traits with higher discriminatory power included total motility, progressive motility, amplitude of lateral head displacement and cytoplasmatic droplets. Through multivariate discriminant analysis, more than 80% of the 140 ejaculates were correctly classified into their own group, showing that this analysis may be an efficient statistical tool to improve the discrimination of potential fertility of boars. Nevertheless, the validation of the relationship between fertility and semen traits using this statistical approach needs to be performed on a larger number of farms and with a greater number of boars.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

提高牛细管冻精精子活力的试验研究

本文从细管冻精的3个关键技术环节研究了稀释液的种类对精子活力与存活的影响,确定了细管冻精精子在液氮冷冻度过冰晶期对其损伤最小的最佳熏蒸高度（最佳冷冻温度）和最佳熏蒸时间,并且对成品冷冻的细管冻精不同解冻温度及时间进行研究。由柠檬酸钠和果糖组成的稀释液精子存活时间长,Tris稀释液冷冻后精子活力高于其他配方;牛细管冻精最佳熏蒸距离为2cm,时间影响不显著,7~9min均可;用50℃温水15s解冻精子的活力最高。     

Relationship of Sperm Quality to Fertility after 4 Days of Cooled Storage of Equine Semen

This study evaluated measures of sperm quality in relation to fertility achieved with fresh semen or semen cooled and stored. Semen from 1 stallion was collected and processed to provide 3 treatments: group 1 received fresh semen; group 2 received cooled semen containing 50% seminal plasma (SP) stored for 4 days; and group 3 received cooled semen containing 50% SP stored for 1 day, then centrifuged and resuspended in fresh extender containing 10% SP on days 1 to 3. Inseminates were evaluated for sperm motion characteristics and the percentage of sperm with intact membranes (SMI). Mares (n = 34) in estrus were treated with an ovulation-inducing drug and inseminated with 100 million membrane-intact sperm on the following day. Pregnancy status was determined via transrectal ultrasonography 2 weeks after ovulation. The mean percentage of SMI was higher in group 1 (81%, initial) than in group 2 (74%, day 4) or group 3 (74%, day 4) (P < .05). The median percentages of total sperm motility differed among the groups (77%, 5%, 59% for groups 1, 2, and 3 respectively; P < .05). Median values for the percentages of progressively motile sperm and curvilinear velocity for group 1 (55%, 216 μm/s) and 3 (37%, 186 μm/s) were higher than for group 2 (1%, 73 μm/s) (P < .05). Pregnancy rates did not differ among groups (5 of 11, 45% in group 1; 5 of 11, 45% in group 2; and 7 of 12, 58%, in group 3; P = .77). These data suggest that, at least for this stallion, sperm membrane integrity may be a more valuable means of assessing potential fertility of cooled-stored semen than sperm motion characteristics.     

冻存液添加蜂王浆主蛋白对精子品质的影响

目的 精液冷冻保存作为人工授精不可或缺的一个技术环节,对优质畜禽种群的繁衍和保存有着至关重要的意义。目前,因精液冻存时冻存液成分、冷冻方法和外部氧化应激等因素影响,导致冻存精液品质不一。蜂王浆（RJ）已被证明能提高动物精液品质,蜂王浆主蛋白（MRJPs）作为RJ主要生物活性成分物质,具有多种生物活性和抗氧化能力。方法 为提高冻存精子品质,本研究开展了在公牛精液冻存液中添加不同浓度（0 g/25 mL、0.01 g/25 mL、0.02 g/25 mL、0.03 g/25 mL、0.04 g/25 mL）的MRJPs冻干粉,对冻存48 h后解冻精子的总活力、前进性活力、畸形率等相关参数进行了观察评估。结果 添加MRJPs呈浓度依赖性方式显著降低精子总活力、快速前进性活力、缓慢前进性活力和直行性活力（P<0.05）,而且精子畸形率和精子原地移动活力与对照组相比无显著差异（P> 0.05）。结论 精液冻存液中添加MRJPs会抑制冻存精子活力,因此,对MRJPs能以何种方式提高精液品质的研究还需要进一步开展。