Comparison of Computer-assisted Sperm Motility Analysis Parameters in Semen from Belgian Blue and Holstein–Friesian Bulls

Subjective microscopic sperm motility results have recently been demonstrated to differ between Holstein-Friesian (HF) and Belgian Blue (BB) bulls. However, such assessments are rather imprecise. In the present study, sperm motility was assessed objectively by means of the Hamilton Thorne CEROS version 12.2c computer-assisted sperm motility analyser (CASA), and differences between the BB and HF breed could also be demonstrated. Higher percentages of both totally (p < 0.0001) and progressively (p < 0.0001) motile spermatozoa were encountered in the HF breed compared with the BB breed. Furthermore, a lower kinetic efficiency of the BB spermatozoa, evidenced by a lower beat cross-frequency (p = 0.0007) combined with a higher lateral head displacement (p = 0.0015), was the basis for the lower velocity of BB sperm cells. Additionally, BB spermatozoa move less straight forward, resulting in a lower straightness (p < 0.0001). No sperm motility differences were observed between age groups within the BB breed. The breed differences were observed in the examined bull populations residing at AI centres, in Belgium for the BB bulls and in the Netherlands for the HF bulls. However, these bull populations are selected for fertility. A similar pattern was observed in an unselected bull population of both breeds, although these differences were mostly non-significant for the different CASA parameters. Nevertheless, these data suggest that a genetic component might be responsible for the observed sperm motility breed differences.     

广州地区娟姗公牛与荷斯坦公牛精液品质的比较研究

本试验选择亚热带气候条件下广州地区的娟姗公牛和荷斯坦公牛各5头,比较两个品种公牛的精液品质(采精量、原精密度、原精活力、细管精液产量、冻后活力、低渗膨胀率及穿透率)。研究表明,荷斯坦公牛每次采精的采精量(16.14±0.06 mL)和细管精液产量(189.17±3.11支)都极显著地高于娟姗公牛(4.74±0.05 mL,158.46±2.64支)(P<0.01);娟姗公牛的原精密度(8.95±0.08亿/mL)极显著地高于荷斯坦公牛(8.32±0.07亿/mL;P<0.01);娟姗公牛原精活力(0.731±0.004)高于荷斯坦公牛(0.729±0.003),但两者差异不显著(P<0.05);娟姗公牛精液的冻后活力(0.355±0.003)极显著高于荷斯坦公牛(0.339±0.003;P<0.01);娟姗公牛冷冻精液的低渗膨胀率(34.50%±0.49%)显著高于荷斯坦公牛(31.21%±0.59%;P<0.01);娟姗公牛冷冻精液对去透明带仓鼠卵的穿透率(84.51%±13.83%)显著高于荷斯坦公牛(81.52%±6.13%;P<0.05)。     

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.     

Storage of Fresh Bovine Semen in a Diluent Based on the Ionic Composition of Cauda Epididymal Plasma

For artificial insemination (AI) in cattle, much lower insemination doses can be applied when fresh semen is used instead of frozen-thawed semen. However, a particular disadvantage of fresh semen is its limited shelf life. As bovine spermatozoa can be stored for several weeks in the cauda epididymis without negative effects on their fertilizing capacity, it is an interesting organ to serve as a model in order to prolong the shelf life of fresh semen. First, the storage capacity of a diluent [cauda epididymal plasma (CEP-1)] with the same ionic composition, pH and osmolarity as the bovine CEP was compared with a Tris diluent for extended preservation of fresh ejaculated bovine semen. Secondly, the ionic composition of the CEP-1 diluent was modified (CEP-2) and its storage capacity was compared with this of the CEP-1 and Tris diluent. Finally, the effect of addition of different polyols (sorbitol, glycerol, mannitol) and egg yolk concentrations (5, 10 and 20%) to the CEP-2 diluent was assessed. Sperm quality decreased rapidly in the CEP-1 diluent. The quality and especially progressive motility of spermatozoa stored in the CEP-2 diluent were better those in the CEP-1 and Tris diluent. No significant effects of different sugars or egg yolk concentrations on the quality of fresh bovine semen in the CEP-2 diluent were observed. In conclusion, the CEP-2 diluent with 10% egg yolk and 1 g/l sorbitol may be used for extended preservation of fresh bovine semen at 5 degrees C up to 6 days.     

Assessment of Sperm Morphology in Zebu Bulls, under Field Conditions in the Tropics

Sperm morphology was studied in 302 extensively managed Zebu bulls (aged 1.5–9 years), classified as sound (n=166) or unsound (n=136) for breeding, under field conditions in the dry tropics of Costa Rica. Single semen samples were collected by electro‐ejaculation and fixed in formol‐saline solution immediately after collection. Sperm morphology was determined in the field on wet smears using a microscope equipped with phase‐contrast optics, and further determined in the laboratory on air‐dried smears stained with carbol‐fuchsin. The frequencies of sperm abnormalities (such as abnormal acrosome, head, neck, mid‐piece, tail, and presence of cytoplasmic droplets) were recorded as a percentage of the total number of counted spermatozoa (400 cells). Zebu bulls considered unsound for breeding showed a higher mean prevalence (p < 0.05) of knobbed acrosomes (4.0 versus 0.9%), head defects [specifically, nuclear invaginations and heads with abnormal shapes and sizes (27.6 versus 4.0%)], abnormal tails (11.2 versus 4.7%), and proximal droplets (8.4 versus 1.6%), compared with bulls considered sound for breeding. In these latter bulls, the abnormality most commonly seen was the presence of single bent tails with an entrapped cytoplasmic droplet (3.0 ± 3.7%). Young Zebu bulls (i.e. bulls under 2 years of age) showed a higher percentage of missing acrosomes, and proximal cytoplasmic droplets, than older sires (12.1 versus 2.4%, and 23.9 versus 3.6%, respectively; p < 0.05), interpreted as an indication of low ejaculation frequency and sexual immaturity, respectively. Bulls with a long scrotum and soft testicular consistency (TC) at palpation showed higher percentages of abnormal sperm heads in the ejaculate than bulls with a normal scrotal length (SL) and a normal TC (32.7 versus 12.8% and 30.7 versus 10.3%, respectively; p < 0.05). In addition, Zebu bulls with a scrotal circumference (SC) ≤ 30 cm showed a higher prevalence of proximal cytoplasmic droplets than bulls whose SC was > 30 cm (9.8 versus 2.6%, p < 0.05). A higher mean percentage of abnormally sized and shaped heads, especially undeveloped and narrow at the base, was more frequently found in stained smears than in unstained samples (26.0 versus 9.9%, p < 0.05), which clearly underlines the importance of using both stained and wet smears when assessing sperm head morphology. However, for a quick assessment of sperm morphology under field, tropical conditions, phase‐contrast microscopy provides useful information for the spermiogramme evaluation.     

The Relationship between Semen Quality and the Nonreturn Rate of Bulls

Contents
The value of routine evaluation of bull semen was analysed for 117 AI bulls placed in two studs. Data from semen analysis of a total of 1635 ejaculates was compared statistically with the nonreturn rates of the bulls. The semen parameters which were significantly correlated to nonreturn rates were the motility of the freshly collected ejaculates (p = 0.0140) and post-thaw motility (p = 0.0075). The total number of motile sperm in the inseminate ranged from 10.9 to 19.3 × 10⁶ and according to previous reports the effects of low motility should be fully 'compensated' when doses above 10 × 10⁶ sperm/dose are used for insemination. In conclusion, the motility of freshly collected semen does not appear to be 'compensation' and a low percentage of motile sperm in an ejaculate may indicate other dysfunctions of the population of motile cells. Furthermore, post-thaw motility appears to correlate significantly with nonreturn rates. The largest proportion of the variation was explained by the breed of the bull and stud (42.2% of the variation), whereas the two motility parameters explained 10% of the total variation in nonreturn rates. Objective and precise evaluation of sperm motility in combination with other semen traits are needed to improve breeding efficiency. Although microscopic evaluation of sperm motility correlates with nonreturn rates of bulls, the methods are subjectively assessed and inaccurate and therefore do not allow a satisfactory prediction of fertility.     

Motility of Fresh and Frozen-Thawed Stallion Sperm from Three Segments of the Epididymal Cauda and the Effect of Post-Thaw Seminal Plasma Addition on Motility

Preservation of epididymal spermatozoa is an advantageous method to preserve genetic material of endangered species or valuable breeding animals after sudden death and injuries. Despite lower pregnancy rates, fertilization with epididymal sperm has been proven successful. Variable sperm quality after cryopreservation among individual stallions and the usually terminal chance to preserve epididymal sperm are opportunities for the development of a freezing procedure suitable for the majority of stallions. To evaluate the effect of the preservation procedure, we analyzed the sperm motion characteristics after every step of processing. In addition, we investigated the influence of seminal plasma on motility of frozen-thawed semen. We compared three segments of the cauda epididymidis and harvested spermatozoa by retrograde flushing (most caudal part) and mincing (more cranial segments) to augment the number of spermatozoa. During processing, there were differences in sperm motion characteristics depending on the segment of the cauda epididymidis. Distinct increases in motility due to different extenders and the effect of seminal plasma suggest that motion characteristics of raw and frozen-thawed spermatozoa are strongly influenced by microenvironment and must be interpreted with caution.     

Comparison of the Effects of Four Freezing Methods on Motility Characteristics,Morphology, and Viability of Postthaw Stallion Epididymal Sperm

Semen cryopreservation is of growing interest in the horse breeding industry, and collecting epididymal sperm might provide the chance to preserve genetic material from valuable stallions after severe injury or death. In case of an unexpected emergency, there may not always be an adequate laboratory nearby. Therefore, we compared fast and slow freezing methods using either a programmable freezer or a styrofoam box filled with liquid nitrogen. Epididymides of 10 stallions were collected immediately after routine castration under general anesthesia. Epididymal spermatozoa were evaluated before and after the freeze-thaw process for motility, viability, morphological, and kinematic parameters. Neither postthaw motility nor kinematic values differed among the four freezing protocols. Morphological abnormalities after freezing and thawing differed among epididymal segments. However, there were significantly more nonviable spermatozoa after the freeze-thaw process using the fast freezing process in the styrofoam box filled with liquid nitrogen compared with all other freezing processes. According to the results of this study, freezing in nitrogen vapor should be considered as an alternative to the programmable freezer only in combination with a prolonged cooling period.     

Comparison between Urine Protein: Creatinine Ratios of Samples Obtained from Dogs in Home and Hospital Settings

Background

The urine protein:creatinine ratio (UPC) is used to quantify urine protein excretion and guide recommendations for monitoring and treatment of proteinuria.

Hypothesis/Objectives

Home urine samples will have lower UPCs than hospital samples. The objectives were to compare UPCs of samples collected in each setting and to determine whether environment of sample collection might affect staging, monitoring or treatment recommendations.

Animals

Twenty‐four client‐owned dogs.

Methods

Prospective, nonmasked study. Clients collected a urine sample from their dog at home and a second sample was collected at the hospital. Dogs receiving corticosteroids or angiotensin‐converting enzyme inhibitors were excluded, as were those with urine samples of inadequate volume, no protein on dipstick analysis, or active urine sediment. Samples were refrigerated after collection, dipstick and sediment evaluations were completed and each sample was frozen at −80°C within 12 hours. UPCs were performed on frozen samples within 2 months.

Results

From 81 paired samples, 57 were excluded. Of the remaining 24, 12/24 (50%) had higher hospital sample UPCs, 9/24 (38%) had identical UPCs, and 3/24 (12%) had lower hospital UPCs. The UPCs of hospital samples were higher than home samples for the total population (P = .005) and the subset with UPC > 0.5 (P = .001).

Conclusions

Setting and related circumstances of urine collection in dogs is associated with UPC differences; results are usually higher in hospital than in home samples. This difference has the potential to affect clinical interpretation.     

Case Study: Relationships of Scrotal Circumference and Scrotal Volume to Growth and Semen Traits in Beef Bulls

Breeding soundness examinations (BSE) were performed on 327 bulls at three locations in Wyoming and Montana. Scrotal circumference (SC), scrotal volume (SV), and body condition score (BCS) data were also collected. The animals were classified as yearlings, 2-yr-olds, or mature bulls. Age class and BCS had significant (P<0.01) effects on SC. Age class also accounted for significant (P<0.01) variation in SV. The correlation between SC and SV was 0.88. Scrotal circumference, SV, and pelvic area (PA) were measured and adjusted for age on the 139 yearling bulls at Location 1 (MT) to allow comparison with other age-adjusted traits. The linear regression of SC on age was 0.023 cm/d (P<0.05). Scrotal circumference and age were significant (P<0.01) sources of variation for the percentage of motile sperm (MOT). Composite yearling bulls had larger (P<0.05) adjusted SV, adjusted SC, pelvic height (PH), and percentage of MOT than Red Angus yearling bulls. The simple correlation between adjusted SC and adjusted yearling BW was 0.33 (P<0.05). Actual SC and SV were positively correlated with actual BW, actual hip height (HH), and percentage of MOT. Scrotal volume and percentage of MOT were positively correlated (0.22) (P<0.05). Our results indicate that SV could be used interchangeably with SC as a measure of sperm- producing capacity in beef bulls. Results of this study indicate that selecting bulls with larger SC or SV should result in increased yearling BW, greater PA, and bulls with improved fertility.     

荷斯坦牛和西门塔尔牛冻精品质及其体外受精后早期胚胎发育的比较研究

为探讨荷斯坦牛和西门塔尔牛冻精的精液品质及体外受精后胚胎发育能力的差异,利用目测法、低渗膨胀法和考马斯亮蓝染色法评估了荷斯坦牛和西门塔尔牛冻精的活力、质膜完整率和顶体完整率,并比较了二者冻精体外受精后胚胎的卵裂率和囊胚率。结果表明,荷斯坦牛和西门塔尔牛冻精的活力(30.4%和27.2%)、质膜完整率(41.96%和36.22%)和顶体完整率(77.02%和73.02%)均无显著差异(P>0.05),但荷斯坦牛冻精体外受精后的卵裂率(57.5%和48.6%)和囊胚率(30.3%和23.2%)显著高于西门塔尔牛冻精(P<0.05)。提示,不同品种公牛精液体外受精后的发育能力有显著差异(P>0.05)。     

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.     

Relationship between Sperm Response to Glycosaminoglycans in vitro and Non-return Rates of Swedish Dairy AI Bulls

In this study, the relations between fertility (56‐day non‐return rates, 56‐day NRR) after artificial insemination (AI) and bull sperm characteristics post‐thaw, after swim‐up and after co‐incubation with heparin (Hep) and hyaluronan (HA), respectively, were determined, attempting to determine if such a procedure could be of value to evaluate the potential fertilizing ability of frozen‐thawed AI bull spermatozoa. Spermatozoa from 20 semen batches derived from 20 Swedish Red and White AI bulls ranging widely in their field fertility after AI (55–79% 56‐day NRRs) were evaluated with regards to post‐thaw motility, membrane integrity, and migration through a simple swim‐up procedure. Sperm viability and capacitation status were evaluated by two different vital staining procedures and chlortetracycline hydrochloride staining. Sperm motility and membrane integrity post‐thaw (e.g. indicators of sperm viability) were significantly correlated (r = 0.53, p < 0.05 and r = 0.59, p < 0.01, respectively) with fertility. Heparin (5 µg/ml) significantly (p lt; 0.001) increased the frequencies of capacitation and acrosome‐reaction (AR) among swim‐up separated spermatozoa, whereas HA at a concentration of 50 ng/ml did not have any significant capacitating effect. The incidences of capacitated or AR‐spermatozoa following Hep‐treatment were not correlated with fertility. On the other hand, the percentage of viable spermatozoa was significantly (p < 0.001) lower in Hep‐treated samples than in control and HA‐treated samples and was significantly (r = 0.49, p < 0.05) correlated with fertility after AI (56‐day NRR). The results indicate that the percentage of viable spermatozoa after swim‐up separation and heparin‐exposure from a selected population of AI bulls were significantly and positively related to the AI fertility of the donors and thus could be used as a parameter to determine the fertilizing ability of frozen—thawed AI bull spermatozoa.     

禄劝推广肉牛冻精改良的经验和体会

禄劝的肉牛冻改技术和冻改头数在均处于云南省领先地位,为禄劝畜牧业发展和贫困山区农民增收致富起到了积极的促进作用,主要经验和体会是领导重视,提高母牛受胎率,良种良法,规范管理、政策扶持和推广留母还犊模式等。     

关于种公牛精液平衡时间与冻后活力关系的研究

本试验的统计分析数据表明，种公牛精液平衡时间与冻后活力存在一定函数关系。     

张掖市肉牛冻精养殖户需求调查报告

根据《张掖市牛产业扶持资金肉牛冻精采购项目代理服务补充协议》要求,因冻精采购的特殊性,需在采购前做好各县区冻精市场需求调查及预判供应商考察等工作。某咨询代理公司于2022年3月3日至16日,聘请专业技术人员组成调查组,选择高台县、民乐县、临泽县、甘州区4个牛产业发展大县,作为张掖市肉牛冻精使用情况和市场需求的调查样本,进行了实地调查。     

不同种公牛X／Y精子分离效果及对体外受精能力的影响

本研究首先时比了3头种公牛的X/Y精子分离效果,并通过常规冻精和性控冻精的活力检测,来评价流式细胞仪分离及冷冻处理对不同种公牛精子分离效果的影响.应用体外受精试验,探讨了不同种公牛个体的常规冻精和分离冻精的受精和胚胎发育能力.试验结果表明,不同种公牛个体间除分离准确率外,精子的分离速率(4.1×103/s vs 4.5×103/s vs5.3×103/s,P<0.05)和死精率(19.3%vs 14.5%vs 11.0%,P<0.05)都存在显著差异.三头种公牛的性控冻精在体外培养过程中,精子活力的下降速率要显著快于常规冷冻精液组.不同种公牛个体的分离冻精组在4小时的精子活力也有显著差异(1号种公牛9.1%vs 2号种公牛22.1%、3号种公牛21.5%,P<0.05).此外,常规冻精和性控冻精在受精后的卵裂率和囊胚率存在显著差异(1号种公牛:64.0%vs 41.6%,26.9%vs 19.3%;2号种公牛:67.3%vs 52.3%,29.2%vs26.5%;3号种公牛:60.3%vs 43.1%,27.3%vs 29.9%).上述结果表明,不同种公牛个体间精子的分离速率、死精率、以及对分离冷冻处理的耐受性存在显著差异,造成其解冻后活力和存活时间不同程度的下降;但在体外受精试验中,3头种公牛的分离精子仍具有正常的受精能力,并能支持胚胎发育到囊胚阶段.综上结果表明,选择适宜的种公牛和降低分离及冷冻处理对精子的损伤,对于提高精子的分离效率、分离精液的品质和体外受精效率都有着重要的意义.     

Effect of Sperm Cryopreservation and Supplementing Semen Doses with Seminal Plasma on the Establishment of a Sperm Reservoir in Gilts

Frozen-thawed (FT) boar sperm have a reduced fertile life, due in part to a capacitation-like status induced by cooling. Reversal of this cryocapacitation in vitro by exposure to boar seminal plasma (SP) has been demonstrated. The objective of these studies was to determine the effect of SP on the ability of FT sperm to create an oviductal sperm reservoir following artificial insemination (AI). In Experiment one, 35 pre-pubertal gilts were injected (IM) with 400 IU eCG plus 200 IU hCG to induce oestrus. At detection of oestrus, gilts were inseminated with 3 x 10(9) live sperm, either fresh (FS; n = 13), FT (n = 10), or FT supplemented with 10% v/v SP (n = 12). Gilts were killed 8 h later, their reproductive tracts recovered and the uterotubal junctions (UTJs) flushed to recover sperm. Fewer (p < 0.01) sperm were recovered following FT, compared to FS, inseminations, and there was no evident effect of SP. In Experiment two, 30 pre-pubertal gilts received IM injections of 1000 IU eCG followed by 5 mg pLH 80 h later to control time of ovulation. Gilts were inseminated with 3 x 10(9) live FS sperm (n = 6), FT sperm (n = 15) or FT sperm plus 10% SP (n = 9) at 12 h before ovulation and then sacrificed 8 h later. The UTJs were dissected and flushed for sperm recovery. Fewest (p < 0.001) sperm were recovered following FT insemination and there was no evident effect of SP. These data demonstrate that the size of the sperm reservoir is markedly reduced in gilts inseminated with FT sperm. However, the lack of effect of SP suggested that either it did not reverse cryocapacitation or that such a reversal does not impact the in vivo ability to create a sperm reservoir.