Comparison of Apoptotic Cells Between Cryopreserved Ejaculated Sperm and Epididymal Sperm in Stallions

The development of a reliable technique to freeze epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The aim of this study was to compare the apoptotic indices of sperm obtained from ejaculate, sperm recently recovered from the epididymides (EP), and sperm recovered from epididymides stored at 5°C for 24 hours (EP-stored). For the first category, two ejaculates from seven stallions were collected and then submitted to cryopreservation using an egg yolk-based extender. One week after the last semen collection, the stallions were submitted to bilateral orchiectomy, and sperm from one of the cauda epididymis was harvested immediately after castration (EP). The remaining testicle was stored in a passive refrigeration container at 5°C for 24 hours before the cauda epididymal sperm was harvested (EP-stored). Sperm harvesting from the epididymis for EP and EP-stored was performed by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender. The recovered sperm was then cryopreserved using the egg yolk-based extender. Sperm motility parameters were studied by computer-assisted semen analysis, and apoptosis was estimated by measuring caspase activity and membrane phospholipid translocation using epifluorescence microscopy. The samples were evaluated immediately (0 hour) and 8 hours after thawing. At 0 hour, no differences in sperm parameters were observed among the groups, but after 8 hours, significant statistical differences were observed in sperm motility parameters and plasma membrane integrity among the treatment groups. In addition, viable cells with no apoptotic signs were more prevalent in EP and EP-stored, suggesting that epididymal sperm is less sensitive to the cold shock caused by sperm cryopreservation.     

Bacterial Flora of Semen Collected from Danish Warmblood Stallions by Artificial Vagina

Semen samples were collected from 21 Danish Warmblood stallions by the Colorado artificial vagina (Colorado AV, 14 samples) or by the Missouri artificial vagina (Missouri AV, 7 samples). The semen was examined bacteriologically by direct plating (DP) on blood agar plates, and by plating of semen swabs stored in Stuart’s transport media (TM) at 4°C for 1–4 days. No significant differences were observed between results obtained by DP and cultures of identical TM samples. Of the 21 samples examined, only 1 TM (4.8%) and 2 DP samples (9.5%) were sterile, while the rest yielded a predominantly mixed flora comprising 1 to 4 bacterial genera. The natural flora was dominated by coagulase-negative staphylococci (Staphylococcus lentus, S. capitis, S. haemolyticus, S. xylosus) (16/21 = 76%), coryneforms (11/21 = 52%) and alpha-hemolytic streptococci and lactobacilli (7/21 = 33%). Potential venereal pathogens were isolated from 7 stallions (33%). Beta-hemolytic streptococci were found in 4 stallions used for natural service, whereas Pseudomonas aeruginosa serotype 6 (2 samples ) and Klebsiella pneumoniae subsp. pneumoniae capsule type K5 (1 sample) were isolated from 3 stallions used exclusively for artificial insemination. The role of the stallion as a carrier of potential venereal pathogens, and the artificial vagina as a source of contamination, is discussed in the context of mare endometritis.     

The Relationship of Mitochondrial Membrane Potential,Reactive Oxygen Species,Adenosine Triphosphate Content,Sperm Plasma Membrane Integrity,and Kinematic Properties in Warmblood Stallions

Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive oxygen species; ROS), ATP content, and plasma membrane integrity were assessed. Motion and functional characteristics differed among investigated stallions (P < .05). In addition, it was revealed MMP was positively correlated with the level of ROS and ATP content and progressive motility (P < .05). The level of ROS was positively correlated with ATP content and negatively correlated with plasma membrane integrity and straightness (P < .05). Adenosine triphosphate content was positively correlated with progressive motility, curvilinear velocity, average path velocity, and beat cross frequency and reversely correlated with plasma membrane integrity and straightness (P < .05). Plasma membrane integrity was positively correlated with straight line velocity, linearity, and straightness and negatively correlated with curvilinear velocity (P < .01). In conclusion, the present study substantiated that kinematic and functional characteristics varied among various warmblood stallions. Furthermore, the present study implicated although higher mitochondrial activity increases ATP synthesis, it leads to elevated superoxide anion production, which could culminate in disintegration of the sperm plasma membrane, thereby altering motion characteristics and swimming pattern of sperm.     

Testicular Measurements and Daily Sperm Output of Tori and Estonian Breed Stallions

Evaluation of testicular measurements and daily sperm output (DSO) yields valuable information for predicting the reproductive capacity of stallions. The present study evaluated testicular measurements (height, length, width and circumference) and DSO of eight Tori and eight Estonian breed stallions. One ejaculate of semen was collected daily for 10 subsequent days from each stallion. The gel‐free volume of semen was measured with a graduated glass cylinder and the sperm concentration was assessed with a Chorjajev chamber. The volume of gel‐free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). The DSO was calculated as mean TSN of collection on days 8–10 in Tori breed stallions and on days 4–10 in Estonian breed stallions. The DSO of Tori breed stallions was 12.9 × 10⁹ spermatozoa and of Estonian breed stallions 4.5 × 10⁹ spermatozoa (p < 0.001). Testicular measurements (in cm) 1 day after the last semen collection were as follows: left testis– height 7.3, length 10.4 and width 7.3 in Tori breed stallions, and 5.9, 8.1 and 5.9, respectively, in Estonian breed stallions; right testis– height 7.4, length 10.6 and width 7.4 in Tori breed stallions, and 5.5, 7.4 and 5.3, respectively, in Estonian breed stallions. All these testicular measurements were significantly smaller in Estonian than in Tori breed stallions (p < 0.001). Testicular circumference was 45.4 and 35.4 cm in Tori and Estonian breed stallions, respectively (p < 0.001). The testicular circumference was correlated with DSO in both Estonian (p < 0.05) and Tori breed stallions (p = 0.071). The results give us valuable information on the reproductive capacity of Tori and Estonian breed stallions.     

Consequences of specialized breeding in the Swedish Warmblood horse population

In many European warmblood studbooks, clear specialization toward either jumping or dressage horses is evident. The Swedish Warmblood (SWB) is also undergoing such specialization, creating a possible need for separate breeding programs and a discipline-specific Young Horse Test (YHT). This study investigated how far specialization of the SWB breed has proceeded and the potential consequences. Individuals in a population of 122,054 SWB horses born between 1980 and 2020 were categorized according to pedigree as jumping (J), dressage (D), allround (AR), or thoroughbred (Th). Data on 8,713 J horses and 6,477 D horses assessed for eight traits in YHT 1999–2020 were used to estimate genetic parameters within and between J and D horses and between different periods. Future scenarios in which young horses are assessed for either jumping or dressage traits at YHT were also analyzed. More than 80% of horses born in 1980–1985 were found to be AR horses, while 92% of horses born in 2016–2020 belonged to a specialized category. The average relationship within J or D category was found to increase during the past decade, whereas the relationship between these categories decreased. Heritability estimates for gait traits were 0.42–0.56 for D horses and 0.25–0.38 for J horses. For jumping traits, heritability estimates were 0.17–0.26 for J horses and 0.10–0.18 for D horses. Genetic correlations between corresponding traits assessed in J and D horses were within the range 0.48–0.81, with a tendency to be lower in the late study period. In the future scenarios, heritability and genetic variance both decreased for traits that were not assessed in all horses, indicating that estimation of breeding value and genetic progress for these traits could be affected by a specialized YHT. However, ranking of sires based on estimated breeding values (EBVs) and accuracy of EBVs was only slightly altered for discipline-specific traits. With continued specialization in SWB, specialization of the YHT should thus be considered.     

Post-thawing Sperm Quality in Chilean Purebred Stallions: Effect of Age and Seasonality

In this work, we investigated the influence of age and seasonality on sperm motility and DNA fragmentation in post-thawing semen from Chilean Purebred Stallions (CPS), a horse breed presenting the oldest genealogy record in South America with an interesting reproductive industry. Despite that semen from aged CPS is frozen all year round, there is a lack of studies characterizing the breed semen freezability in accordance with age and seasonality. Twenty fertile CPS were grouped into the young group, the middle group, and the aged group. Ten ejaculates from each stallion were obtained by using an artificial vagina during summer (December) and winter (July) and directly frozen. Subsequently, the frozen semen was thawed and analyzed by a computer-assisted semen analysis and flow cytometer assessing progressive motility, mean velocity, and DNA fragmentation spermatozoa. Kruskal–Wallis test and Pearson’s correlation were used to determine statistical differences among groups and correlation among variables (P ≤ .05). Both spermatozoa motility traits decreased progressively in accordance with age and seasonality, showing the lowest values in the aged group during winter and the highest values in the young group during summer. Deoxyribonucleic acid fragmentation increased significantly in accordance with age and seasonality being highest in the aged group during winter and lowest in the young group during summer. Post-thawing sperm quality showed a negative correlation with the age of the stallions and a positive correlation with the normal sperm morphology before freezing. These results allow the conclusion that age and seasonality are important factors that need to be considered during the selection of CPS for reproductive programs.     

Radiological investigations of osteochondrosis dissecans in Standardbred Trotters and Swedish Warmblood horses

A total of 106 Standardbred Trotters and 27 Swedish Warmblood horses, with a radiological diagnosis of osteochondrosis dissecans, were studied over a six year period. The majority were young horses. No statistical difference in frequency between the sexes was demonstrated. In both breeds osteochondrosis was most common in the hock joints, the site of predilection being the distal dorsal tip of the intermediate tibial ridge. On radiographs the lesions of the hock joints were graded on a scale from 0 to 5 according to size, number and localisation of defects and visible loose bodies. The sizes of the loose bodies estimated radiologically were fairly closely correlated with those found at surgery or autopsy.     

Influence of foreign stallions on the Swedish Warmblood breed and its genetic evaluation

The objective of this study was threefold: 1) To survey the use and impact of foreign stallions in the Swedish Warmblood (SWB) breed 2) To investigate the impact of including genetic groups in the genetic evaluation of the SWB and 3) To estimate the impact of incomplete pedigree information on the estimated breeding values (EBVs) for stallions. Data consisted of 189,330 horses of which 50,907 had performance information. Studbook of origin was determined for stallions with at least 5 progeny tested in Sweden. Those 757 stallions had together 116,505 progeny registered in the SWB. Genetic groups were formed according to preliminary analyses of the genetic levels in dressage and show jumping of the different populations. During the first studied period, comprising horses born before 1980, about 80% of the foals were sired by Swedish born stallions, whereas these only sired 40% of the horses born 1990–2003. In this period Holstein stallions had sired 21% of the foals and Dutch stallions (KWPN) 10%. The proportion of mares covered by foreign stallions increased after the early 1980s to about 80% in 2006. Some of the foreign populations, e.g., Holstein, Selle Francais and KWPN, have significantly affected the show jumping performance level of the SWB breed in the past although the deviation from SWB stallions has become smaller over time. Regarding dressage, no particular foreign population has consistently shown such an impact on the performance although Oldenburg stallions seem to be influential lately. The pedigree completeness (PEC values) of the horses included amounted on average to 0.845 for horses with performance data and to 0.907 for stallions with at least 15 progeny. Foreign stallions had an average PEC value of 0.858. When simulating no pedigree information available for foreign stallions, the EBVs of these stallions changed on average by 19–22 index units, which equals about one genetic standard deviation. Thus, keeping complete and correct pedigree registers is crucial for the assessment of reliable EBVs of SWB horses. Including genetic groups in the genetic evaluation of the SWB resulted in hardly any re-ranking of horses. Correlations between EBVs were about 0.99. Inclusion of genetic groups caused larger standard errors of the EBVs. Furthermore, as the pedigrees on average have great depth, and partially also because Warmblood sport horses have become a mix of many different populations, it was not recommended to include genetic groups in the model for future routine genetic evaluations of SWB.     

Influence of imported sport horses on the genetic evaluation of Swedish Warmblood stallions

There is substantial international influence on the Swedish Warmblood (SWB) sport horse population. The SWB Association suspects that imported horses are pre-selected based on their anticipated performance level, which could lead to biased estimated breeding values (EBVs) for stallions. This study examined different strategies to handle records for imported horses in the genetic evaluation. The stallions were evaluated for 10 traits using 3 different EBV estimation methods that were compared based on ranking comparisons of stallions, accuracy, correlations and absolute differences between EBVs. The results showed that the stallions’ EBVs were affected by imported offspring which, as a group, had higher average scores than horses born in Sweden. Sire EBVs differed by up to 0.6 genetic standard deviation of the trait between methods for stallions with >50% imported offspring. Excluding imported offspring lowered the accuracies and caused larger re-ranking compared with including a fixed effect of origin in the model.     

Effect of Sperm Cryopreservation on the European Eel Sperm Viability and Spermatozoa Morphology

The main objective of the present work was to study the effect of cryopreservation of European eel sperm both on the sperm viability and the spermatozoa head morphology. Spermatozoa morphology was evaluated with computer-assisted morphology analysis after collection in fresh samples, after adding the freezing medium containing dimethyl sulfoxide as cryoprotectant and, finally, after the cryopreservation process and thawing. Cell viability was assessed, in both fresh and thawed samples, by Hoechst 33258 staining. Computer-assisted sperm analysis (CASA) was used to determine the percentage of motile cells and to measure motility parameters in sperm samples. A significant decrease of head perimeter (12.56%) and area (17.90%) was detected from spermatozoa in fresh to thawed samples, indicating that cells do not recover the original size after the cryopreservation process. CASA was used to measure the percentage of motile cells (51.9%) and spermatozoa motility parameters such as curvilinear, straight line and angular path velocities, as well as beating cross frequency. This technique was employed in the fresh sperm samples but proteins present at the freezing medium (L-alpha-phosphatidylcholine) made impossible to use this last technique in thawed samples. When sperm viability was assessed by Hoechst staining, a significant decrease of approximately 15% (73.10 vs 58.26%) of alive spermatozoa was registered from fresh to thawed samples. The percentage of motile cells measured by CASA in fresh samples (51.9%) was lower than the percentage of alive cells determined by Hoechst stainning, suggesting the existence of different batches of spermatozoa in different stages of development, even during the eight to tenth weeks of treatment, when the highest sperm quality was found.     

Genetic correlations between similar traits in the Danish and Swedish Warmblood sport horse populations

Genetic correlations between phenotypically similar or related traits tested at young horse performance tests for Danish Warmblood (DWB) and Swedish Warmblood (SWB) horses were calculated using Multi-trait Across Country Evaluation (MACE). Data comprised stallions with an estimated breeding value (EBV) from the national genetic evaluations (NGE) based on at least 10 progeny tested in performance tests, and the ancestors of those stallions in two generations. The DWB data included 349 stallions and the SWB data 426 stallions. Of these, 28 had EBVs in both DWB and SWB. Additionally 151 pedigree animals were common between DWB and SWB. The dependent variables used were NGE results of stallions born 1980 and later, which reduced the number of common stallions with EBVs to 23. The genetic correlations were very high for jumping traits (0.99) and dressage related traits (0.89–0.97). For conformation traits correlations varied between 0.10 and 0.98. Because of the high genetic correlations and frequent use of same or closely related foreign stallions, breeders of both DWB and SWB would benefit from using the NGEs for performance traits across countries, although the genetic correlations do not consider differences in genetic merit levels between the populations. It would be feasible to perform a joint genetic evaluation using MACE, which would improve the reliability of estimated breeding values, and enable ranking of all stallions according to the national scale of each country.     

Sperm Morphology Assessment in Captive Neotropical Primates

The main objective of this study was to evaluate sperm morphology in four neotropical primate species to compare the sperm morphological traits and the sperm morphometric parameters as a basis for establishing normative sperm standards for each species. Data from 80 ejaculates collected from four primate species, Callithrix jacchus, Callimico goeldii, Alouatta caraya and Ateles geoffroyi, were analysed for detection of sperm morphological alterations using subjective World Health Organization (WHO‐2010) standards and Sperm Deformity Index (SDI) criteria, objective computer‐assisted sperm morphometry analysis (CASMA) and subpopulation sperm determination (SSD) methods. There were multiple differences (p < 0.01) observed among primate species in values obtained from WHO‐2010, SDI, CASMA and SSD sperm analysis methods. In addition, multiple significant positive and negative correlations were observed between the sperm morphological traits (SDI, Sperm Deformity Index Head Defects, Sperm Deformity Index Midpiece Defects, Sperm Deformity Index Tail Defects, Normal Sperm, Head Defects, Midpiece Defects and Tail Defects) and the sperm morphometric parameters (SSD, Area (A), Perimeter (P), Length (L), Width (W), Ellipticity, Elongation and Rugosity) (p ≤ 0.046). In conclusion, our findings using different evaluation methods indicate that pronounced sperm morphological variation exists among these four neotropical primate species. Because of the strong relationship observed among morphological and morphometric parameters, these results suggest that application of objective analysis methods could substantially improve the reliability of comparative studies and help to establish valid normative sperm values for neotropical primates.     

Use of field records and competition results in genetic evaluation of station performance tested Swedish Warmblood stallions

The main objective of this study was to investigate possibilities to increase accuracy in the selection and test of Swedish warmblood stallions by combining information from stallion performance tests (SPT) with information from competitions and riding horse quality tests (RHQT). Data on 801 stallions judged at the SPT 1979–2005 were used for the analyses, which also included about 14 900 horses from the RHQT, 26 800 horses with results in show jumping and 14 200 horses with results in dressage competitions.Heritability coefficients were on average 0.41 for individual gaits under rider and 0.36–0.65 for jumping traits in SPT. Heritabilities for competition traits were 0.17 for dressage and 0.27 for show jumping. The heritability for overall conformation in SPT was 0.25. Genetic correlations between stallion performance test traits and competition results were 0.44–0.77 for gaits and dressage, and 0.78–0.96 for jumping traits in SPT and show jumping. The genetic correlation between conformation and results in dressage competitions was 0.22.For stallions participating in SPT in 2004 and 2005 accuracy increased when evaluating stallions by adding information from relatives that have taken part in RHQT and competitions. For show jumping accuracy changed from 0.60 to 0.68 for stallions born in Sweden, an increase with 13%, when information from RHQT and competitions in show jumping were used. For stallions born in a foreign country, the corresponding figure was, 5%, lower. For dressage the information from RHQT and competition results in dressage changed the accuracy from 0.41 to 0.55, an increase with 34%, for stallions born in Sweden. The improvement in accuracy for imported stallions was 11%.     

马鹿精子形态与超微结构研究

文中对马鹿(Cervus elaphus)精子形态与超微结构进行了研究。马鹿精子经固定、脱水、置换、包埋和聚合,用超薄切片机切片,再用醋酸双氧铀、柠檬酸铅染片,最后于透射电镜下观察其超微结构的变化。观察结果表明:马鹿精子全长(58.75±2.35)μm,由头部(8.93±0.24)μm、颈部(1.00±0.16)μm和尾部(48.18±1.18)μm三部分组成;头部呈扁卵圆状,绝大部分被浓缩且电子密度较高的精核所占据,顶体似帽状扣在精核之上,约占头部的2/3,其前部较为膨大;颈部位于头部和尾部之间,这个区域较短;尾部可分为中段、主段和终段。马鹿尾部轴丝的结构类型为"9+2";微管结构类型为"9+9+2"。     

四种活体染色法评定家禽精子形态的研究

精子形态学检测是预测精子功能最有价值的指标之一,形态异常精子越多受精率越低。用4种伊红-苯胺黑活体染色法(Blom、Bakst、Morisson、Jaskowski)评价三黄鸡和贵妃鸡的精子形态,结果表明:染色方法显著影响精子形态,但不影响精子活率,染液对精子自然形态的保持至关重要。Blom染色法导致精子头部膨胀,畸形精子显著增多(P≤0.01),Bakst、Morisson和Jaskowski染色法均可作为评定家禽精子形态的实用方法,其中Jaskowski染色方法最简便经济,值得在家禽生产中推广应用。