Effect of pasteurized egg yolk on the quality of cryopreserved boar semen

Egg yolk (EY, control) is an essential ingredient of diluents for boar semen cryopreservation. Pasteurized egg yolk (PEY) reduces hygienic risks in processing and is easier to standardize. The aim of this study was to evaluate the in vitro effect of PEY (treatment) on frozen-thawed boar semen. In a split-sample approach (n = 13 boars), it could be shown that there is neither an influence (p > .05) on post-thawing motility (PTM: 5, 30 and 120 min) nor on morphologically intact sperm, percentage of acrosome defects and membrane fluidity using a PEY extender compared to the control. Mitochondrial activity (p = .043), membrane integrity (p = .015) and PTM 300 min (p = .023) were slightly affected in the treatment group. Overall, sperm quality was at a high level in both experimental groups. Further studies are needed to determine the impact of PEY on the fertilizing capacity of boar ejaculates.     

Effect of selenium and vitamin E addition to the extender on liquid stored capercaillie (Tetrao urogallus) semen quality

Captive breeding has become an important tool in species conservations programmes, maintaining genetic diversity and restoring wild, endangered populations. In order to improve the reproductive efficiency of captive kept capercaillie, the purpose of the study was to determine the effect of selenium and vitamin E addition to semen extender on sperm characteristic during short‐term storage. Ejaculates collected individually from four capercaillie were divided into two parts, diluted threefold with basic EK extender and EK enriched with 1 mg/ml of organic selenium and 8 mg/ml of vitamin E (EK+Se+E) and stored 24 hr at temp. +4°C. Spermatozoa morphology, motility and motility parameter were evaluated in net, diluted and stored semen samples. Significant (p < .05) differences between individual males were stated in relation to the majority of traits evaluated in the freshly collected semen. Comparing to the fresh semen, a significant (p < .05) decrease in percentage of live sperm in total (by 3.8% points on average) has been observed in samples diluted by EK extender, while in semen diluted with EK+Se+E extender this decrease was lower (1.5%pts on average) and not significant. Also per cent of motile sperm in EK+Se+E extender was higher (p < .05) then in EK (71.6% vs. 58.9%), but taking into account the values of individual males, both extender and male effect on liquid semen storage become apparent. Obtained data allow concluding that selenium and vitamin E addition to EK extender had positive effect on morphology and motility of capercaillie semen stored 24 hr at 4°C and can be recommended for similar studies carried out on other Galliformes species.     

Semen quality improvement in boars fed with supplemental wolfberry (Lycium barbarum)

Wolfberry is well known for its health benefits in Asian countries. This study consisted of two experiments. In Experiment 1, nine boars were provided 40 g dried wolfberry per 100 kg body weight per day in addition to regular feed for 160 days (divided into 40 days phases: I, II, III, and IV) under step‐down air temperature conditions. Controls (n = 9) were fed regular feed only. Significant (p < .05 or p < .01) or slight improvements in sperm progressive motility, total abnormality rate, sperm concentration, and total sperm per ejaculate were observed in the wolfberry group during phases II and III. No differences were observed in semen volume. After combining the data from phases II ~ IV, significant improvements were detected in all aforementioned traits (p < .05 or p < .01), except semen volume. In Experiment 2, the wolfberry group (n = 5) was fed wolfberry for 90 days and exhibited significantly reduced head, tail, and total abnormality rates (p < .05 or p < .01) in both fresh semen and semen stored for 72 hr at 17°C compared to the control group (n = 5). SOD activity also significantly increased in this group of boars. Collectively, the findings of this study suggest that wolfberry has a positive effect on boar semen quality.     

An approach to rescue the fertility of stallions with a high level of hemospermia

A high amount of blood and not the mere presence of blood in equine semen impacts fertility. The aim of this study was to develop an approach to rescue the fertility of stallions with high hemospermia levels. Semen from 15 stallions was divided into four experimental groups: (a) Control—pure raw semen, (b) WB50—50% (v/v) whole blood added into semen, (c) E1—WB50 extended in a 1:1 (v/v) ratio with milk-based extender and (d) E2—WB50 extended in a 2:1 ratio with milk-based extender. Sperm kinetics, plasma membrane integrity (PMI), lipid peroxidation (PER) and intracellular superoxide (O₂) production were immediately evaluated. Four cycles of 20 mares were randomly assigned to the experimental groups. Mares were bred with an insemination dose of 1 billion total sperm and pregnancy was diagnosed 14 days after ovulation. Sperm kinetics could not be evaluated in the WB50 samples. Total motility was lower (p < .05) in E1 than in CT and E2 samples. Progressive motility decreased (p < .05) with an increase in the percentage of blood in the samples. The PMI and PER did not differ between groups (p > .05); however, O₂ production was higher (p < .05) in WB50 than in E2 samples, while the values were intermediate (p > .05) for CT and E1 samples. The control (90%) and E2 (90%) groups had superior (p < .05) fertility than the others (WB50—0% and E1—25%). It was concluded that sperm motility and fertility of semen with a large amount of blood can be rescued by dilution with a 2:1 extender:semen ratio using a milk-based extender.     

Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio^®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio^® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio^® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio^® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio^® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio^® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.     

Comparative effect of slow and rapid freezing on sperm functional attributes and oxidative stress parameters of goat spermatozoa cryopreserved with tiger nut milk extender

Comparative effect of slow and rapid freezing on sperm functional attributes and oxidative stress parameters of goat spermatozoa cryopreserved with tiger nut milk (TNM) extender was examined in this study. Pooled semen samples obtained from West African Dwarf (WAD) goat bucks were diluted with Tris‐based extenders containing different levels of TNM (0, 5, 10, 15 and 20 ml/100 ml extender). The diluted semen samples were subjected to slow and rapid freezing for a period of 7 days and thereafter evaluated for sperm functional attributes (percentage motility, acrosome integrity, membrane integrity, abnormality and livability) and oxidative stress (malondialdehyde [MDA] concentration and acrosin activity) parameters. Results showed that higher (p < 0.05) motility, livability, membrane and acrosome integrities in semen cryopreserved with slow freezing compared to rapid freezing. These parameters (motility, livability and membrane integrity) were higher (p < 0.05) in semen cryopreserved with 15% TNM in both slow and rapid freezing protocols. The results revealed that semen cryopreserved in slow freezing had lower (p < 0.05) abnormality compared to rapid freezing. Acrosin activity was higher in slow freezing compared to rapid freezing. Acrosin activity was higher at 15% TNM in both slow and rapid freezing. Lower (p < 0.05) MDA concentration was observed in semen cryopreserved using slow freezing compared to rapid freezing. The findings revealed improved post‐thaw sperm functional attributes and oxidative stress parameters of WAD goat spermatozoa cryopreserved with 15% TNM using slow freezing.     

Purification of cryopreserved camel spermatozoa following protease-based semen liquefaction by lectin-functionalized DNA-defrag magnetic nanoparticles

Although incorporating proteases into sperm medium is considered the most effective procedure to eliminate camel semen viscosity, it drastically affects viability, morpho-functional properties and, hence, fertilization potential of spermatozoa. The present work aimed at evaluating adequacy of employing magnetic nanoparticles-based sperm purification technique for eluting impaired and apoptotic camel spermatozoa from cryopreserved semen doses following protease-based semen liquefaction. Thirty cryopreserved semen doses (50 x 10⁶ sperm/straw) representing the following liquefaction treatments: control (untreated), 0.1 mg/ml papain or 5 U/ml bromelain were used (n = 10 straws per treatment). Immediately after thawing (38°C for 40 s), sperm concentration of each straw within treatment was readjusted to 15 x 10⁶ sperm/mL by dilution in PBS (37°C). Sperm physical and cytological properties were then assessed (non-purified semen). Thereafter, each specimen was subjected to lectin-functionalized DNA-defrag magnetic nanoparticles sperm purification, and the same sperm traits were re-evaluated after undergoing purification (purified semen). Sperm DNA fragmentation level within each group, prior to and after magnetic nano-purification, was also determined by fluorescent imaging. The results showed a dramatic improvement (p < .05) in post-thaw motility (%), viability (%), normal sperm (%), intact acrosome (%) and HOST-reacted (%) spermatozoa in protease-liquefied semen following sperm magnetic nano-purification. Additionally, the highest (p < .05) DNA fragmentation level was recorded in all cryopreserved semen groups prior to purification, whereas the lowest (p < .05) was observed in the protease-treated specimens after magnetic nano-purification. These results indicate that protease-based semen liquefaction prior to cryopreservation in conjunction with magnetic nano-purification post-thawing holds potential for reducing the proportion of damaged and dead spermatozoa, hence improving camel sperm fertilization competence.     

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1-hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane-intact spermatozoa determined by computer-assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen-thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.     

Variation among stallions in sperm quality after single layer centrifugation

Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48 hr using computer-assisted sperm analysis and flow cytometry. Sperm quality was better in SLC than in CON at all timepoints, especially chromatin integrity and MI (p < .0001 for both), and some categories of ROS production (e.g. proportion of live hydrogen peroxide negative spermatozoa, p < .0001), but the degree of improvement varied among stallions and type of ROS (p < .05–p < .0001). Total and progressive motility were also better in SLC samples than in CON at 24 and 48 hr (p < .0001), although the effect on sperm kinematics varied. The interaction of treatment, time and stallion was not significant. In conclusion, sperm quality was better in SLC samples than in CON, although there was considerable individual variation among stallions. The improvement in sperm quality, particularly in chromatin integrity, was clearly beneficial, and therefore the use of this technique would be warranted for all stallion semen samples.     

Sperm subpopulations influence the pregnancy rates in cattle

This study aimed to study the characteristics and subpopulations of spermatozoa from bulls with low and high reproductive performance based on pregnancy rates. Based on historical records of pregnancy rate from four farms, 24 bulls were selected. Two groups were established, with low pregnancy rates (n = 12; LOW), including bulls that presented pregnancy rates <52.27% (33.33% to 51.81%); and a group with high pregnancy rates (n = 12; HIGH), with pregnancy rates >52.27% (52.27% to 69.64%), after fixed-time artificial insemination (FTAI). The thawed sperm straws were analysed to sperm kinetics, morphology, plasma membrane integrity and sperm subpopulations. The LOW group exhibited a higher proportion of static cells (p < .05). In contrast, the HIGH group showed greater percentages for membrane integrity and total and progressive motility, and cells with fast and medium velocity (p < .05). In the cluster procedures, four sperm subpopulations were established. The low-fertility bulls presented the highest percentage of subpopulation 2 (41.46%), characterized by slow and progressive spermatozoa. The high-fertility bulls exhibited the highest percentage of subpopulation 3 (37.17%), characterized by fast and nonlinear spermatozoa. Results from this study indicated that bulls with greater percentages of fast and nonlinear spermatozoa seem to have greater fertilization capacity and the subpopulations analysis can be considered a tool to identify ejaculates with high fertility.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

Irradiation of semen doses with LED‐based red light in a photo chamber does not improve in vitro quality of thermically stressed boar spermatozoa

Recent reports indicate that stimulation of liquid‐stored boar semen with red LED‐based light improves sperm quality and reproductive performance in sow herds. So far, in vitro data after LED stimulation of whole semen doses are lacking. In this study, the effect of LED light exposure on the in vitro quality of boar spermatozoa after storage and thermic incubation was examined. Boar semen doses were stored at 17°C (n = 10) or 5°C (n = 6) in Beltsville Thawing Solution extender and then exposed to red LED light using a commercial photo chamber. During a subsequent long‐term incubation at 38°C, neither sperm kinematic parameters nor mitochondria function or membrane integrity differed between control and treated samples (p > .05). It is concluded that stimulation of semen doses in the LED‐photo chamber does not improve quality of thermically stressed boar sperm in vitro. Other than the sperm traits tested here might be involved in the previously reported improvement of in vivo fertility.     

Intracellular calcium chelating agent (BAPTA‐AM) aids stallion semen cooling and freezing–thawing

This study aimed to investigate the effects of different concentrations of 1,2‐bis‐(o‐aminophenoxy)‐ethane‐N,N,N0 N0‐tetraacetic acid, tetra‐acetoxymethyl ester (BAPTA‐AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing–thawing. After collection, semen was extended (1:1 v/v) on a skim milk‐based extender, centrifuged and resuspended at 400 million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200 μΜ BAPTA‐AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120 hr and after freezing–thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved‐thawed semen. Cooled stored (48 hr) semen containing 50 μΜ BAPTA‐AM and control extender (0 μΜ BAPTA‐AM) was used to assess fertility. Inclusion of 50 μΜ BAPTA‐AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p < 0.05). Furthermore, semen cryopreserved in extender containing 50 μΜ BAPTA‐AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p < 0.05). Addition of 50 μΜ BAPTA‐AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p > 0.05). In conclusion, the addition of BAPTA‐AM to semen extenders aided stallion semen cryopreservation in a dose‐dependent manner. Furthermore, the cooling extender supplemented with 50 μΜ BAPTA‐AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA‐AM.     

Antioxidative effect of BHA in soya bean lecithin‐based extender containing Glycerol or DMSO on freezing capacity of goat semen

The aim of this study was to evaluate the effects of butylated hydroxyanisole (0 or 4 mM) along with different concentrations (5 or 7%) of glycerol (G) and dimethyl sulphoxide (DMSO) as cryoprotectant (CPAs) on freezability of goat semen. Semen was collected from four bucks (3–4 years) twice a week for five weeks. The pooled ejaculates were diluted with extender containing two different concentrations of G or DMSO in combination with BHA. Afterwards, the diluted samples were loaded into 0.25 ml straws and frozen using a standard protocol. After thawing motility parameters, viability, membrane integrity and total abnormality were assessed. The Results showed that the presence of BHA in extender, type and level of CPAs as main factors had significant effects on goat sperm viability, total and progressive motility after freezing–thawing processes (p < .05). Also, the interaction of BHA (0 and 4 mM) and levels of G or DMSO (5 or 7%) had a significant effects (p < .05) on total motility, viability and some characteristic. In this case, the addition of 5% G or DMSO with BHA resulted in highest motility and viability than the other groups (p < .05). The addition of G5 (with and without BHA) increased VSL and reduced abnormality than the other groups (p < .05). The results showed that the main effects of CPAs and CPAs level on membrane functionality were significant (p < .05). Also there were no significance differences in the interactive effects of MDA, VCL, VAP, ALH, LIN and STR among the groups (p > .05). Finally, it can be concluded that the use of 5% CPAs with or without BHA may result in better post‐thaw sperm quality of goat.     

Influence of dietary crude protein content on fattening performance and nitrogen excretion of Holstein steers

The objective was to investigate the influence of crude protein (CP) content in a fattening diet on feed intake, body weight gain, nitrogen excretion, and carcass traits in Holstein steers. Steers (initial body weight 241 ± 26 kg) consumed feed with the following CP content: (a) 17.7% during the early period (from 7 to 10 months of age) and 13.9% during the late period (from 11 to 18 months of age) (HIGH, n = 3), and (b) 16.2% during the early period and 12.2% during the late period (LOW, n = 4). The CP intake was lower in the LOW than the HIGH group. Urinary and total nitrogen excretion in the late period tended to be lower (p < .10) in the LOW than the HIGH group. However, growth performance and carcass traits were not affected by dietary CP content. Free histidine and total amino acid contents in the longissimus thoracis muscle tended to be higher (p < .10) in the HIGH than the LOW group, however, the CP contents were not affected by dietary CP content. The results of this experiment suggest that decreasing dietary CP to 16% (early period) or 12% (late period) of dry matter would reduce nitrogen excretion from Holstein fattening farms without affecting productivity.     

Estimation of genetic parameters and season effects for semen traits in three pig breeds of South China

The economic profitability of a boar station largely depends on semen quantity and quality traits. However, genetic analysis of semen traits has not yet been done in the boar population in China. In this study, we aimed to estimate genetic parameters for semen traits and the influence of seasons on these traits by using data of Duroc, Landrace and Yorkshire boars in South China. The following four semen traits were analysed: semen volume (ml; VOL), sperm concentration (10⁶/ml; DEN), sperm motility (MOT) and percentage of abnormal sperm (ABN). Genetic parameters and season effects were estimated simultaneously for each breed by using a multiple‐trait (4 × 4) repeatability animal model. The four traits had a moderate heritability with average estimates of 0.23, 0.28, 0.26 and 0.17 across the three breeds, respectively. The estimates of genetic correlations among four traits differed in the three breeds. In particular, in Yorkshire, the four traits were nearly genetically independent. The season of collecting semen had a significant impact on these four semen traits except ABN in Duroc (Bonferroni adjusted p < 0.05/6). The moderate heritabilities indicate the possibility of effective selection of boars for semen traits. Different genetic correlations for the three breeds suggest that the selection strategy for the four traits should be investigated separately for each breed. Some necessary actions should be taken to reduce the influence of seasons on semen traits.     

Adjusted method of penis fixation during boar semi-automatic semen collection aiming to reduce bacterial contamination

Semen collection has an essential role in the initial bacterial load in boar ejaculates and extended semen. The study aimed to explore the efficacy of an adjusted penis fixation in a semi-automatic collection system on reducing bacterial contamination of ejaculates in two-boar studs with different scenarios. Historically, stud A had low levels of bacterial load in raw semen, while stud B had a high level of contamination. A total of 56 mature boars had their semen collected using two methods of penis fixation: (a) Traditional: The penis was fixed directly with the artificial cervix and transferred to the adjustable clamp; (b) Adjusted: The fixation was performed with one gloved-hand, and after exteriorization, the penis was gripped using the artificial cervix with the other gloved-hand and transferred to the adjustable clamp. The bacterial load (p = .0045) and the occurrence of ejaculates >231 CFU/ml (p = .0101) were reduced in the Adjusted compared to the Traditional method. Bacterial load was reduced when using the Adjusted method in stud B (p = .0011), which showed a greater occurrence of critical factors for bacterial contamination (p ≤ .0034). The Adjusted method reduced the occurrence of ejaculates >231 CFU/ml when the preputial ostium was dirty (p = .016) and the duration of semen collection was >7 min (p = .022) compared to the Traditional method. In conclusion, the Adjusted penis fixation was efficient in reducing bacterial load of ejaculates, mainly in boar stud B, which had high contamination challenges.     

Insemination of sows with seminal doses prepared by a two-step hypothermic dilution does not impair the reproductive performance at farm

Reproduction in swine is mostly carried out through artificial insemination (AI). For this purpose, AI studs collect the ejaculates, analyse the sperm quality, dilute and package to produce seminal doses and ship them to sow farms to carry out the AI. Temperature is controlled during the process to avoid sperm damage. Semen is diluted in the extender in a one-step or a two-step process where the second can be isothermic (approximately 32°C) or hypothermic (room temperature 21–22°C). Both techniques are currently performed, and the latter could reduce time and costs, but the literature available comparing the processes is scarce and presents discrepancies. To date, there are no studies about its impact in fertility. This study compared hypothermic two-step dilution (HTSD) and isothermic two-step dilution (ITSD) in laboratory and field trial to elucidate whether HTSD has any effect. Ejaculates from 72 boars in nine AI studs were split and processed with both techniques using a high-performance extender and evaluated in laboratory. Four farms inseminated 345 sows with samples of four of these AI studs, and their fertility and prolificacy were registered. Results show no significant differences between doses prepared by HTSD and ITSD technique, having no impact in laboratory results (percentage of motile sperm, short hypoosmotic swelling test (sHOST) and short osmotic resistance test (sORT), viable sperm, damaged acrosomes, sperm under early apoptosis, high mitochondrial membrane potential (p > .1), fertility (92.2% versus 94.1%, p = .45) or farrowing rate (15.8 ± 0.3 versus 16.1 ± 0.3 p = .46). In conclusion, our results suggest that HTSD of semen on extender could be safely implemented in AI studs under the conditions tested.     

Evaluation of a portable device for assessment of motility in stallion semen

In horse breeding, quality assessment of semen before insemination is often requested. Non‐laboratory‐based techniques for objective analysis of sperm motility are thus of interest. The aim of this study was evaluating a portable device for semen analysis (Ongo sperm test) and its comparison with computer‐assisted semen analysis (CASA). Semen was collected from 10 stallions, diluted to 100, 50 and 25 × 10⁶ sperm/ml and analysed for total (TM) and progressive motility (PM). The final sperm concentration influenced total motility analysed by Ongo (p < 0.05) which was higher at 100 × 10⁶ sperm/ml when compared to 25 × 10⁶ sperm/ml (p < 0.05) but not when compared to 50 × 10⁶ sperm/ml (n.s.). Sperm concentration did not influence total motility when assessed by SpermVision (n.s.). Agreement between methods was evaluated by correlation analysis and Bland–Altman plot. Intra‐assay variation of Ongo was 5.2% ± 3.0 for TM and 6.9% ± 3.4 for PM. Correlation between Ongo and CASA was r = 0.79, 0.88 and 0.83 for 100, 50 and 25 × 10⁶ sperm/ml for TM, and r = 0.87, 0.89 and 0.87 for PM, respectively (all p < 0.001). At the 100 and 25 mio/ml dilutions, the difference between the two systems deviated significantly from 0, while no such bias existed at the 50 mio/ml dilution (TM Ongo 85.0%, CASA 82.3%; PM Ongo 64.1%, CASA 66.1%). The 95% confidence interval was 19.9%, 18.9% and 19.2% ± mean for TM and 20.7%, 17.4% and 20.3% ± mean for 100, 50 and 25 × 10⁶ sperm/ml, respectively. In conclusion, Ongo sperm test sperm motility data were strongly correlated with data obtained by CASA. In addition, at a concentration of 50 × 10⁶ sperm/ml values measured with both systems were close to identical. At this concentration, which is recommended in equine AI, Ongo and CASA can be used interchangeably.