Lateral Head Displacement Research Articles

A combination of biomarkers for predicting stallion sperm fertility

Equine breeding would benefit greatly from reliable biomarkers of stallion or ejaculate fertility. The aim of the study was to investigate how several in vitro sperm characteristics correlate with fertility after artificial insemination, to explore the potential to build a fertility prediction model for stallions. Cooled insemination doses (3–5 per stallion) were obtained from various studs. Sperm membrane integrity, acrosome integrity, chromatin integrity, mitochondrial membrane potential, and reactive oxygen species production were evaluated by flow cytometry 24–30 h after semen collection, and sperm motility was assessed by computer aided sperm analysis. Calcein violet was used to differentiate viable spermatozoa. Per season pregnancy rates for these stallions were available the following year. Positive correlations were found between pregnancy rate and straightness (r = 0.43, p ≤ 0.001), as well as pregnancy rate and the proportion of living hydrogen peroxide positive spermatozoa (r = 0.32, p ≤ 0.05). There were negative correlations between pregnancy rate and amplitude of lateral head displacement (r = -0.26, p ≤ 0.05), and between pregnancy rate and the mean fluorescence of dead superoxide positive spermatozoa (r = -0.46, p < 0.001). Principal component analysis indicated that motility, membrane integrity, DNA fragmentation, and reactive oxygen species production were associated with pregnancy rate. Therefore, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates. However, data from more individuals would be required to construct a model for fertility prediction.

ProAKAP4 as Indicator of Long-Lasting Motility Marker in Post-Thaw Conditions in Stallions.

ProAKAP4, a precursor of AKAP4 (A-kinase anchor protein) found in the flagellum of mammalian and non-mammalian spermatozoa, serves as a structural protein with established correlations to motility parameters across diverse species. This study aimed to determine the proAKAP4 level evolution in thawed stallion semen over a 3 h period, examining its correlation with motility descriptors and mitochondrial membrane potential. Utilizing sixteen ejaculates from four French warmblood stallions, this study involved maintaining thawed samples at 37 °C for 3 h, conducting proAKAP4 enzyme-linked immunosorbent assays (ELISA), computer-assisted sperm analysis (CASA), and mitochondrial membrane potential by JC-1 probe and flow cytometry at 0, 1, and 3 h post-thawing. The findings indicate significant positive correlations (p ≤ 0.05) between proAKAP4 levels and sperm total or progressive motility at all time points analyzed. Spermatozoa velocity descriptors (VAP, VCL, VSL) and spermatozoa lateral head displacement (ALH) display positive correlations (p ≤ 0.05) with ProAKAP4 at the 0 h post-thawing. ProAKAP4 concentration exhibits no discernible difference between batches with or without a cryoprotectant. Notably, proAKAP4 consumption remains insignificant within the initial hour after thawing but becomes significant (p ≤ 0.05) between 1 and 3 h post-thawing. In summary, proAKAP4 demonstrates positive correlations with total and progressive motility in stallion semen for up to 3 h after thawing, albeit showing a noticeable decrease starting from the first hour post-thawing, indicating a progressive consumption as a result of spermatozoa motile activity.

L-carnitine enhances the kinematics and protects the sperm membranes of chilled and frozen-thawed Peruvian Paso horse spermatozoa

l-carnitine (LC) transports fatty acids to the mitochondria for energy production, reducing lipid availability for peroxidation through β-oxidation. This research examines the effect of LC supplementation to two skimmed milk-based extenders on the cryosurvival of chilled (5°C) and frozen-thawed Peruvian Paso horse spermatozoa .An initial experiment determined the optimal LC concentration (0, 1, 5, 10, 25, and 50 mM) when added to INRA-96® and UHT (skimmed milk + 6% egg yolk) extenders, using nine ejaculates from three stallions chilled for up to 96 h. Subsequently, the effect of 25 mM LC supplementation (the optimal concentration) on chilling (INRA-96) and freezing (INRA-Freeze®) extenders was evaluated using eight pooled samples from sixteen ejaculates (2 ejaculates/pool) from four stallions. Results indicated that all LC concentrations produced significantly higher values (P<0.05) for kinematic variables (total [TM] and progressive motilities, curvilinear [VCL] and straight-line [VSL] velocity, and beat-cross frequency [BCF]), and the integrity of plasma/acrosome membranes (IPIA) compared to non-supplemented chilled sperm samples for up to 96 h with both extenders. Moreover, the use of 25 mM LC was more efficient (P<0.05) in preserving the post-chilled values of velocity, BCF, and IPIA for the long term than lower LC concentrations (1–10 mM). Post-thaw values of total motility, the amplitude of lateral head displacement (ALH), and IPIA were significantly improved (P<0.05) when INRA-Freeze extender was supplemented with 25 mM LC. In conclusion, supplementation of l-carnitine to skimmed milk-based extenders enhanced kinematic variables and protected the membrane integrity in chilled and frozen-thawed Peruvian Paso horse spermatozoa.

Comparative developmental competence of in vitro embryos recovered from Bali cattle with normal and poor sperm motility.

Fertility is crucial for enhancing the efficiency of livestock production, as it directly impacts the reproductive rates. A comprehensive understanding of the relationship between sperm quality and embryo development is key to optimizing reproductive outcomes and improving the quality of livestock. This study analyzed the developmental competence of in vitro embryos recovered from Bali cattle with normal or poor sperm motility. Nine bulls with normal fresh semen (NFS) or poor fresh semen (PFS) motility were ejaculated for semen. Semen ejaculates, including volume, motility, and sperm concentration, were evaluated immediately after collection to measure the quality of the fresh semen. Frozen semen was evaluated using computer-assisted semen analysis (CASA) for motility, progressive sperm motility, distance curve path, distance curve linear, distance straight line, average path velocity, curvilinear velocity, linear velocity, straightness (STR), linearity of forward progression (LIN), wobble, and average lateral head displacement (ALH). Bull groups were used to determine in vitro embryo cleavage ability after fertilization of Bali cattle. Ovaries of Bali cattle were collected by slicing, and only cytoplasmic oocytes with compact cumulus cells were used in this study. The oocytes were matured, and in vitro fertilization was performed using fertilization media with a final sperm concentration of 1.5 × 106 spermatozoa/mL. After 48 h, the embryo cleavage ability of the cultured oocytes was evaluated. There were significant differences in motility values between the NFS and PFS groups; however, there were no significant differences in the volume or sperm concentration. There was a significant difference in the LIN value between the groups but no significant differences in other CASA parameters. There were no significant differences in the cleavage rate and morula between the groups, but a positive correlation was observed between the cleavage rate and the morula and between the morula and ALH. A significant negative correlation was observed between the cleavage rate and STR and between the morula and STR; no significant differences were observed for other variables. Despite variations in sperm characteristics, both normal and poor sperm motility demonstrated comparable in vitro embryonic development competence. These findings provide important insights into the fertility potential of Bali bulls, providing valuable information that can enhance selection strategies to improve the quality of livestock production.

Effect of quercetin, L-ergothioneine and H89 on sperm motility and kinematic pattern, plasma membrane functionality and in vitro heterologous fertilizing capacity of cryopreserved equine semen

Semen cryopreservation causes extensive chemical and physical damage to sperm structure, which generates premature aging and reduces viability and fertility of spermatozoa. The addition of antioxidants to freezing extenders can reduce the oxidative damage caused by excessive generation of reactive oxygen species (ROS), and the premature aging could be reduced by adding an enzyme inhibitor that prevents an anticipated capacitation. The aim of this study was to evaluate the in vitro effect of quercetin (Q), L-ergothioneine (E) and H89 addition to cryopreserved equine spermatozoa. Six experimental groups were stablished: control, Q, E, H89, H89Q and H89E. The analyzed parameters were sperm motility and kinematic using computer assisted sperm analysis (CASA), plasma membrane functionality with the hypoosmotic swelling test (HOST) and fertilizing capability with in vitro heterologous fertilization. Quercetin reduced curvilinear velocity (VCL) and increased beat-cross frequency (BCF), while its combination with H89 (H89Q) reduced total motility, progressive motility, VCL and hyperactive sperm (HA). Likewise, H89 and its combination with E (H89E) decreased VCL and amplitude of lateral head displacement (ALH). No significant differences were observed among treatments for membrane functionality and fertilizing capacity of sperm. In conclusion H89 in combination with Q and E reduced sperm motility or some kinematic parameters. However, they did not influence plasma membrane functionality and in vitro fertilizing capacity of frozen-thawed equine semen.

Protein factors of capacitation and decapacitation in canine seminal plasma

The aim of the present study was to determine the proteins in canine seminal plasma with direct effect on specific sperm parameters during fertilisation. The influence of seminal plasma proteins was focused on three processes: capacitation, hyperactivation and decapacitation. Size exclusion chromatographic fractional separation of proteins from seminal plasma based on their molecular weights was performed. In addition, two-dimensional electrophoresis of two seminal protein fractions was done. The effect of protein molecules on the motion and kinetic parameters of the male gametes in in vitro conditions was monitored by computer-assisted sperm analysis. The activity of alkaline phosphatase, creatine kinase and alanine aminotransferase in whole seminal plasma and each chromatography fraction was measured spectrophotometrically using the BA-88A semi-automatic chemistry analyzer. The protein fractions with different molecular weights affected the in vitro motility and kinetic characteristics (curvilinear velocity, straightness, linearity and amplitude of lateral head displacement) of spermatozoa, leading to hyperactivation or initiating decapacitation kinetics changes. The detailed study of the seminal plasma proteome would add fundamental information about the processes associated with physiological changes occurring in spermatozoa before fertilisation. The study of the canine seminal plasma proteome could add relevant information about its effects on the fertilising ability of the male gametes and the changes occurring in them before fertilisation.

Model test and finite element analysis results of a monopile in very dense sand under unidirectional horizontal cyclic loading

This study is carried out to understand the response of a monopile for a large (8–9 MW) offshore wind turbine located at a 45m water depth under lateral cyclic loading. Firstly, a 1g large-scale model pile test was performed in saturated very dense sand under numerous cycles of lateral loading, N=2000. The test results show that the lateral pile head displacement and rotation increase while the secant stiffness of pile decreases with increasing N. The numerical model was then validated by comparing the pile head displacement results of the test and the finite element (FE) model analysis. The soil was modelled using the UBC3D-PLM constitutive model to understand the development of the excess pore pressure ratio (EPPR) around the monopile. The FE analysis was performed cycle by cycle up to N=5 for a large diameter “XL monopile” with diameter D=9m and embedded length L=50m. The results showed that the EPPR around the monopile is insignificant. Furthermore, the effect of the number of loading cycles on the evolution of the EPPR, pile head deformation and pile head rotation are insignificant. In addition, a relatively high maximum pile head rotation accumulation θmax≅0.13° was obtained during cyclic loading.

Effects of semen quality on pregnancy rate in artificially inseminated dairy cows

The study aimed to evaluate the quality of fresh and frozen-thawed semen of five adult Holstein-Friesian crossbred bulls and the pregnancy rate of cows inseminated with frozen semen of those bulls. The fresh semen of breeding bulls collected for artificial insemination (AI) programme in the field was of good quality with volume (6.7 ± 0.2 ml - 8.9 ± 0.5 ml), concentration (904.2 ± 56.4 million/ml), mass activity (3.3 ± 0.2-3.6 ± 0.2), total motility (77.0 ± 1.1% – 92.1 ± 0.6%), progressive motility (67.0 ± 1.2% – 87.4 ± 0.6%) and semen viability (73.0 ± 0.6% to 85.4 ± 0.7%). The computer assisted sperm analysis (CASA) results showed that diluted pre-freezing semen had good sperm total motility (50.1 ± 3.8 % to 59.0 ± 4.7%), progressive motility (30.0 ± 1.2%-39.0 ± 1.6%), the velocity traits of straight linear velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP) of sperm ranged from 48.0 ± 1.3 - 71.3 ± 0.7 μm/s,118.1 ± 2.8-181.3 ± 10.9 μm/s and 68.4 ± 2.5 to 91.0 ± 2.9 μm/s, respectively. Bull 1 showed significantly higher VSL (71.3 ± 0.7 μm/s), VCL (181.3 ± 10.9 μm/s) and VAP (91.0 ± 2.9 μm/s) compared to others. Viability of frozen-thawed semen was lower in Bull 5 (73.0 ± 1.71%) compared to others. Although in frozen-thawed semen these parameters declined, the semen was sufficiently good to be used in AI in the field. The overall pregnancy rate using frozen semen was 55.6% and the highest pregnancy rate (62%) was in cows that were inseminated with frozen semen of Bull 1, but the differences between bulls was not significant. The pregnancy rate had positive correlation with sperm count, total motility, progressive motility, VCL, VSL, VAP, amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), straightness (STR), sperm viability. In artificially inseminated cows, the intensity of oestrus of cows, timing of AI, site of semen deposition and season had a significant effect on pregnancy rate. In conclusion, the fresh and frozen-thawed semen of breeding bulls supplied in North-East Bangladesh for AI programme were good quality. Heat detection and insemination timing need to be improved to increase the pregnancy rate.&#x0D; Bangl. vet. 2022. Vol. 39, No. 1-2, 1–15

Discriminant Analysis and Data Mining CHAID Decision Tree as Tools to Evaluate the Buffering Effect of Hydroxytyrosol on Reactive Oxygen Species in Rooster Sperm Cryopreservation.

Sperm cryopreservation is effective in safeguarding genetic biodiversity in avian species. However, during this process, spermatozoa are very susceptible to plasma membrane peroxidation in the presence of high concentrations of reactive oxygen species (ROS). To mitigate this effect, the addition of exogenous antioxidants, such as hydroxytyrosol (3,4-dihydroxyphenylethanol; HT), an antioxidant derived from olive oil, to the cryopreservation sperm diluent, could be useful. To verify this, a cryopreservation diluent was supplemented with different concentrations (0 μg/mL, 50 μg/mL, 100 μg/mL, and 150 μg/mL) of HT. For this, semen was collected in 10 replicates from 16 roosters of the Utrerana avian breed, and a pool was prepared with the optimum quality ejaculates in each replicate. After cryopreservation, spermatozoa were thawed and different in vitro semen quality parameters were evaluated. A discriminant canonical analysis (DCA) was carried out and revealed that total motility (TM; Lambda = 0.301, F = 26,173), hypo-osmotic swelling test (HOST; Lambda = 0.338, F = 22,065), and amplitude of lateral head displacement (ALH, Lambda = 0.442; F = 14,180) were the variables with the highest discriminant power. Finally, a chi-squared automatic interaction detection (CHAID) decision tree (DT) was performed excluding fresh semen samples and ROS was found to be the most valuable variable to discriminate between the different established freezing groups. Samples in the absence of HT or with low concentrations of this antioxidant showed less desirable ROS values in cryopreserved rooster semen. The present study could lead to the improvement of cryopreservation techniques for the genetic material of local poultry breeds and optimize the conservation programs of endangered native avian breeds.

Evaluation of postnatal growth, hematology, telomere length and semen attributes of multiple clones and re-clone of superior buffalo breeding bulls

The present study comprehensively evaluates the postnatal growth, hematology, telomere length, and semen attributes of multiple clones and re-clone derived from superior buffalo breeding bulls. To the best of our knowledge, we successfully produced multiple clones and a re-clone of an earlier cloned buffalo bull from an embryo developed from an adult bull's skin-derived cell for the first time. The cloned bulls' growth, blood hematology, plasma biochemistry, and telomere length were all shown to be normal at various stages of development. The bulls were used for semen production after being screened for testicular growth and training. Semen characteristics such as volume, concentration, and initial motility of fresh sperm as well as motility and kinetics characteristics such as straightness (STR), average lateral head displacement (ALH), and beat cross frequency (BCF) of frozen-thawed sperms of the cloned bulls were found to be similar to those of non-cloned bulls, including the donor bulls. Additionally, it was found that cloned bulls' functional sperm attributes, including acrosome intactness, mitochondrial membrane potential, and superoxide anion status, were comparable to those of non-cloned bulls. These characteristics are necessary for sperm to pass through the female reproductive system, penetrate the oocyte, and efficiently fertilize. Finally, this study adds to our understanding of the postnatal development, hematology, telomere length, and sperm characteristics of superior buffalo breeding bulls that have been cloned and re-cloned. The findings provide the groundwork for improving cloning practices, refining reproductive procedures, and optimizing the use of cloned genetic material in animal breeding and conservation.

Low-density lipoproteins, resveratrol and quercetin as alternative additives to improve boar semen cooling.

Low-density lipoproteins (LDL), quercetin (Q) and resveratrol (R) have been used for sperm preservation to improve sperm quality in different species. To evaluate the effects of LDL, Q and R during the cooling of boar semen. Fifteen boar semen samples were diluted in a BTS extender supplemented with the treatments: LDL at 6%, Q at 10 μM (Q10), 30 μM (Q30) and 50 μM (Q50), or R at 10 μM (R10), 30 μM (R30) and 50 μM. A control without supplementation was included. The semen was stored by cooling at 16°C for 96 h. Every 24 h, sperm motility and kinetics were evaluated using a computer-assisted sperm analyzer (IVOS). At 24 and 96 h of cooling, functional membrane integrity and mitochondrial membrane potential (ΔΨM) of sperm were evaluated by the hypoosmotic swelling test (HOST) an flow cytometry with JC-1 probe, respectively, LDL improved progressive motility of sperm during cooling. Likewise, LDL increased average path velocity (VAP) and straight-line velocity (VSL) and/or curvilinear velocity (VCL) during the first 48 h of cooling. The use of Q between 10 and 30 μM caused a reduction in total motility, progressive motility and amplitude of the lateral head displacement during the entire cooling period, as well as a decrease in VAP, VSL and VCL at 96 h of cooling. LDL, Q10, Q30 and Q50 modulated mitochondrial activity by reducing high-ΔΨM sperm at 0 and 96 h of cooling. During the cooling of the boar semen prior to artificial insemination, the parameters of sperm quality that could influence fertility decrease; however, the inclusion of antioxidants and additives that protect the plasma membrane, such as LDL, could mitigate the damaging effects on spermatozoa. It is concluded that LDL can improve the motility and kinetics of boar semen during cooling while it could modulating the sperm's mitochondrial activity. On the contrary, Q could alter the motility and kinetics of boar sperm during the cooling period.

Comparative evaluation of Duragen® and skim milk extenders to enhance ram semen quality and fertility: A promising alternative in ovine artificial insemination.

This study aimed to investigate the effectiveness of Duragen® and skimmed milk (SM) extenders on the quality parameters, bacterial load and fertilization ability of stored ram semen. A total of 50 ejaculates from Sardi rams (n = 5) aged 2.5-3 years, were collected and stored in Duragen® and SM at 15°C. The motilities and velocity parameters generated by the CASA system were then evaluated at 0, 8 and 24 h of storage. Afterward, bacterial loads of sperm extended in Duragen® and SM were determined at 0, 5 and 24 h of incubation. In addition, ewes (n = 100) aged 2 years, have been chosen in the same herd. The selected ewes were then synchronized and inseminated using semen extended in Duragen® and SM and stored for 5 h at 15°C. The results revealed that total and progressive motilities, straight velocity (VSL), straightness (SRT), lateral head displacement (ALH) and beat cross frequency (BCF) were not affected by the extender type after 24 h of storage (p > .05). However, curvilinear velocity (VCL), velocity average path (VAP), linearity (LIN) and wobble (WOB) showed higher values in Duragen® compared with SM extender after 24 h of storage (p < .05). Bacterial loads were observed mainly in sperm stored in SM at 5 h (183 UFC/mL) and at 24 h (357 UFC/mL) of incubation. However, the only case showing a bacterial load in Duragen® is when the storage time attains 24 h (199 UFC/mL). Concerning fertility, sperm diluted in both extenders resulting in high fertility rates which reaches 66% and 73% for Duragen® and SM, respectively, with no statistical difference (p > .05). In summary, Duragen® extender decreased bacterial load in stored semen and maintained high ram sperm quality and fertility. These findings suggest that Duragen® extender could be used as SM alternative in ovine artificial insemination (OAI).

Effects of CatSper Stimulation and Inhibition by Progesterone and NNC on Human Sperm

Calcium ion enters the sperm through a specific calcium channel, CatSper. This voltage-sensitive channel is stimulated by intracellular alkalization and progesterone. This study is aimed at investigating the effects of CatSper inhibition or stimulation on sperm motility, viability, and sperm function regulators such as mitochondrial membrane potential (MMP), ATP, and reactive oxygen species (ROS) production. This study was performed on 30 semen samples of fertile volunteers, referred to Shiraz Fertility Center. The semen samples were diluted to 20 × 10 6 sperm/mL. The samples were divided randomly into control, solvent, progesterone (10 μM), NNC (2 μM), and NNC+progesterone groups. Sperm kinematics, viability, MMP, ATP content, and the amount of ROS production were assessed using VT-SPERM3.1, eosin staining, JC1 flow cytometry, bioluminescence, and chemiluminescence methods, respectively. Sperm viability and total and progressive motility were significantly decreased in the NNC and NNC+progesterone groups. The amplitude of lateral head displacement (ALH) and curvilinear velocity (VCL) was reduced in the NNC-containing groups. These parameters did not change in the progesterone group. ROS production by viable spermatozoa in the NNC and NNC+progesterone groups was significantly higher than the controls. MMP and ATP content did not show any significant difference with controls in none of the experimental groups. NNC inhibits the CatSper and reduces human sperm motility and viability. These harmful NNC effects were not due to their impact on MMP or ATP production but are likely because of intracellular calcium reduction and higher ROS production. Progesterone at 10 μM concentration had no significant effect and may not be a considerable stimulator for CatSper.

P-090 The in vitro motility characteristics of human sperm collected by microfluidic device

Abstract Study question Do the in vitro motility characteristics of sperm collected using a microfluidic device (ZyMot) differ from those of sperm collected by density gradient centrifugation (DGC)? Summary answer ZyMot-collected sperm had longer-lasting motility than those collected by DGC, but the head beat-cross frequency was inferior due to the absence of washing in vitro. What is known already ZyMot (DxNow, Inc., USA) can sort highly motile sperm with low reactive oxygen species (ROS), and DNA damage, without centrifugation. In IUI cycles, although the total number of ZyMot collected sperm was lower than that of DGC, it was reported that the pregnancy rate of ZyMot cycles was higher than that of DGC cycles. Washing sperm with a medium is essential for in vitro capacitation; however, the protocol for microfluidic devices lacks this procedure. The aim of this study was to clarify the motility characteristics of ZyMot-collected sperm. We also examined how washing ZyMot-collected sperm affected motility. Study design, size, duration The specimens were provided by patients who underwent semen analysis between October 2022 and January 2023, with written consent. We used 12 samples with a semen volume of 2.5 ml or more, or a motile sperm concentration of 4 million/ml or more. The same donor samples were divided into three groups (DGC, ZyMot, and ZyMot with wash). Dunnett’s test was performed with the DGC group as the control group. Participants/materials, setting, methods In the DGC group, 2-layer DGC (400G, 20 min) was performed, followed by washing (400G, 5 min). In the ZyMot group, ZyMot was used according to the manufacturer’s protocol. In the ZyMot with wash group, ZyMot-collected sperm was centrifugally washed (300G, 5 min). All samples were cultured at 30 °C until motility analysis. Sperm motility was analyzed using a computer-assisted sperm analysis system (SMAS) after 0, 4, and 24 h of semen preparation. Main results and the role of chance There was no significant difference in the motility rate of the two ZyMot groups compared to that of the DGC group at 0 and 4h after sperm preparation. On the other hand, the motility of the ZyMot group tended to be higher (53.3%, p = 0.0787), and the motility of the ZyMot with wash group was significantly higher (85.4%, p &amp;lt; 0.001) than that of the DGC group (33.5%), after 24h of sperm preparation. The straight-line velocity, curvilinear velocity, and average speed of the ZyMot and ZyMot with wash group did not differ from those of the DGC group at 0 and 4h. All speed parameters of the ZyMot with wash group were significantly higher than those of the DGC group, after 24h of culturing. Regarding sperm head movement, there was no significant difference in the amplitude of lateral head displacement between the two ZyMot groups compared to that of the DGC group at 0 and 4h of culture. In contrast, the sperm head beat-cross frequency of the ZyMot group was lower than that of the DGC group (9.18±0.31 Hz vs 8.71±0.49 Hz, p = 0.0409). Both head movement parameters of the ZyMot with wash group were significantly higher than those of the DGC group. Limitations, reasons for caution We chose semen samples with a high number of motile sperm because we divided the sperm into three experimental groups. Therefore, it is unclear whether our findings are applicable to patients with poor semen parameters. Wider implications of the findings ZyMot may be advantageous for IUI cycles because ZyMot-collected sperm maintains sustainable motility for extended periods. Alternately, ZyMot may be unfavorable compared to DGC in conventional IVF cycles due to inferior sperm head movement, which is a marker of capacitation. However, this disadvantage can be overcome by washing. Trial registration number not applicable

Effect of bovine seminal plasma Binder of Sperm Protein 1 on stallion sperm cryopreservation

Proteins present in seminal plasma directly interact with the plasma membrane of spermatozoa, being important for successful sperm maturation and fertilization. The aim of this work was to evaluate the effect of Binder of Sperm Protein 1 (BSP1) from the seminal vesicle of bulls on semen quality of stallions and its effect on freezability of this semen. The BSP1 protein was isolated using the ammonium sulfate technique and lyophilized. Protein presence was observed at the highest concentration of BSP1 (98%) confirmed by SDS-PAGE and Western blot. The effect of addition of BSP1 to stallion semen was tested at concentrations 0 (control), 10, 20 and 40 μg/mL by addition to a commercial freezing extender. Semen from 4 stallions with 5 ejaculates per stallions was used and cryopreserved using a programmable machine (TK 3000®). After thawing (37°C / 30 seconds), samples were submitted to sperm kinematic analysis by CASA (time 0 hour and Thermo Resistance Test - TRT of 1 hour at 37°C), integrity of plasma membranes (iMP) and acrosomal membrane integrity (iAC; epifluorescence microscopy. The percentage values were subjected to arcsin transformation and means were compared using T-test at 5% probability comparing time 0 vs. TRT of 1 hour. No difference was observed for iMP andiAC regardless of BSP1 concentration. At time 0h there was a difference at the highest BSP1 concentration (40 μg/mL) (P <0.05) for ALH and BCF (control 2,53 ± 0,08a and 40 μg 2,40 ± 0,07b). After 1h TRT, the concentration of 40 μg / mL continued to present a significant difference. Hyperactivation is characterized by an increase in average lateral head displacement (ALH, mm), and beat cross frequency (BCF, Hz), possibly due to an increase in calcium influx. Our results indicate exactly the opposite with a significant reduction in these two parameters. These results suggest that in the horse BSP1 acts modulating calcium influx. Mitochondrial function determined by JC-1 showed a significant difference at the concentration of 10 μg/mL of the protein, lower than the other groups. As the bovine has a higher fertility compared to stallion, these results, in addition to the novelty of the work, allow further studies using proteins from heterologous species with the aim of improving seminal quality in stallions. In conclusion, BSP1 may exert an antioxidant effect in relation to the mitochondrial potential of sperm and change sperm kinematics due to a modulation of calcium influx by BSP1 protein. Further investigations are required to evaluate intracellular calcium concentrations after addition of BSP1.

The relationship between sperm quality parameters and stallion fertility

There are stallions with apparently good sperm quality that achieve low pregnancy rates when their semen is used for artificial insemination (AI). Thus, additional methods of evaluating sperm quality that result in better methods of predicting stallion fertility, are essential. Semen samples were obtained in April-June from 8 stallions, 4 trotters and 4 warmblood sport horses, which were sampled Monday, Wednesday and Friday each week. From each stallion, 1-4 samples were obtained, and evaluated after cold storage for 24 hours. The samples were evaluated for kinematic variables using CASA, and for membrane integrity, mitochondrial membrane potential (MMP), production of reactive oxygen species, acrosome integrity and chromatin integrity using flow cytometry. Relationships with fertility, calculated as number of pregnant mares/number of inseminated mares for samples used for artificial insemination after transport in cold storage, were evaluated by linear regression, following log-transformation of variables found not to be normally distributed. The following sperm variables were found to have a positive relationship with fertility: Straightness (r2=0.86, p= 0.0009), Viable with High mitosoxred/Low MMP (r2=0.70, p= 0.01), Viable non acrosome reacted (r2=0.68, p= 0.01), Viable by calceinviolet (r2=0.67, p= 0.01), Viable non H2O2-producing (r2=0.66, p= 0.01), Viable with High mitosoxred/High MMP (r2=0.65, p= 0.02), Viable non superoxide-producing (r2=0.60, p= 0.02), Linearity (r2=0.56, p= 0.03). The only variable found to have a negative relationship with fertility was Amplitude of lateral head displacement (r2=0.63, p= 0.02). The other 26 measured variables did not show a significant relationship with fertility. The results of the study shows that both kinematic variables as well as flow cytometric analysis can be valuable for relating fertility with sperm quality in vitro. The flow cytometry results indicate that analysis of the production of different reactive oxygen species can be valuable when predicting stallion fertility. Acknowledgments: Special thanks to Menhammar, Västerbo, Markebäck and Stockholm Seminstation for providing stallion semen. This work was supported by funding from FORMAS (2018-01083; awarded to AJ). The work was also supported by the Swedish University of Agricultural Sciences via the Cells for Life Platform.

Antioxidant effects of zinc-oxide nanoparticles on post-thaw quality and in vivo fertility of Beetal buck spermatozoa

Metallic nanoparticles are generally considered potential cryoprotectants during semen cryopreservation owing to their ability to improve the spermatozoa quality following cryopreservation when added in cryo-diluents. The present study aimed at investigating the effects of incorporating zinc oxide nanoparticles (ZnO-NPs) in the cryo-diluent on in vitro post-thaw sperm quality attributes, oxidant-antioxidant profile, and in vivo fertility rate in Beetal buck. After characterization, ZnO-NPs were added to the Tris-citric acid-fructose yolk extender. Semen samples, collected from five breeding bucks (5 ejaculates per buck) using an artificial vagina, were pooled. The pooled semen samples, distributed into five equal aliquots, were diluted in the Tris-citric acid cryo-diluent containing five different concentrations of ZnO-NPs (0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml, and 0.0 mg/ml called control group), and were cryopreserved for 24 h. At post-thaw, various in vitro sperm quality parameters, and antioxidant enzyme profiles like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and reduced glutathione (GSH) levels were determined. For the in vivo fertility experiment, breeding does (n = 152), aging from 1.5 to 4 years, were artificially inseminated with spermatozoa with intact acrosome containing the same five concentrations of ZnO-NPs. Results demonstrated that the 1.0 mg/ml ZnO-NPs (crystallite size of 19.73 nm) improved (p < 0.01) the activities of antioxidant enzymes (SOD, CAT, and POD) with concomitantly decreased levels (p < 0.001) of lipid peroxidation (LPO) and reactive oxygen species (ROS) when compared with the other groups. Total spermatozoa motility, rapid velocity, and average path velocity were higher (p < 0.05) with the 1.0 mg/ml ZnO-NPs when compared with the control group. Progressive motility and straight-line velocity were higher (p < 0.05) with the 0.5, 1.0, and 1.5 mg/ml of ZnO-NPs addition compared with the 2.0 mg/ml and control groups. The sperm kinematics parameters; the amplitude of the lateral head displacement, the beat cross frequency, the straightness, and the linearity were not influenced by the ZnO-NPs inclusion. Supra-vital plasma membrane integrity was higher (p < 0.001) with the 0.5 mg/ml and 1.0 mg/ml of ZnO-NPs added groups when compared with either 2.0 mg/ml ZnO-NPs or the control group. The viable sperm with intact acrosome was higher (p < 0.05) in the 1.0 mg/ml ZnO-NPs group compared with the other groups. Low supplementation rates improved (p < 0.05) the DNA integrity when compared with the higher dose (2.0 mg/ml ZnO-NPs). The pregnancy per AI was higher (59.3 %; P < 0.05) in the does inseminated with semen added with the 0.5 mg/ml ZnO-NPs followed by 1.0 mg/ml (58.8 %), 1.5 mg/ml (58.6 %), and 2.0 mg/ml (46.6 %) when compared with the control group (33.3 %). We concluded that the 1.0 mg/ml ZnO-NPs added in the citric acid-fructose yolk extender improved the post-thaw quality of Beetal buck spermatozoa in terms of elevated activities of endogenous antioxidant enzymes, improved structural and functional integrity along with higher fertility.

The effect of reclined seatback angles on the motion of booster-seated children during lateral-oblique low-acceleration impacts

Belt-positioning boosters (BPB) may prevent submarining in novel seating configurations such as seats with reclined seatbacks. However, several knowledge gaps in the motion of reclined child occupants remain as previous reclined child studies only examined responses of a child anthropomorphic test device (ATD) and the PIPER finite element (FE) model in frontal impacts. The aim of this study is to investigate the effect of reclined seatback angles and two types of BPBs on the motion of child volunteer occupants in low-acceleration far-side lateral-oblique impacts.Six healthy children (3 males, 3 females, 6–8 years, seated height: 66±3.2 cm, weight: 25.2±3.2 kg) were seated on two types of low-back BPB (standard and lightweight) on a vehicle seat and restrained by a 3-point simulated-integrated seatbelt on a low-acceleration sled. The sled exposed the participants to a low-speed lateral-oblique (80° from frontal) pulse (2 g). Three seatback recline angles (25°, 45°, 60° from vertical) with two BPB (standard and lightweight) were tested. A 10-camera 3D-motion-capture system (Natural Point Inc.) was used to capture peak lateral head and trunk displacements and forward knee-head distance. Three seat-belt load cells (Denton ATD Inc) captured peak seatbelt loads. Electromyography (EMG, Delsys Inc) recorded muscle activation. Repeated Measure 2-way ANOVAs were performed to evaluate the effect of seatback recline angle and BPB on kinematics. Tukey’s post-hoc test for pairwise comparisons was used. P-level was set to 0.05.Peak lateral head and trunk displacement decreased with the increasing seatback recline angle (p < 0.005, p < 0.001, respectively). Lateral peak head displacement was greater in the 25° compared to the 60° condition (p < 0.002) and in the 45° condition compared to the 60° condition (p < 0.04). Lateral peak trunk displacement was greater in the 25° condition than the 45° condition (p < 0.009) and the 60° condition (p < 0.001), and in the 45° condition than the 60° condition (p < 0.03). Overall peak lateral head and trunk displacements and knee-head forward distance were slightly greater in the standard than the lightweight BPB (p < 0.04), however these differences between BPBs were small (∼10 mm).Shoulder belt peak load decreased as the reclined seatback angle increased (p < 0.03): the shoulder belt peak load was statistically greater in the 25° condition than the 60° condition (p < 0.02). Muscle activation from the neck, upper trunk, and lower legs showed great activation. Neck muscles activation increased with the increase in seatback recline angle. Thighs, upper arms, and abdominal muscles showed small activation and no effect of conditions.Child volunteers showed decreased displacement suggesting that reclined seatbacks placed the booster-seated children in a more favorable position within the shoulder belt in a low-acceleration lateral-oblique impact, compared to nominal seatback angles. BPB type seemed to minimally influence the children’s motion: the small differences found may have been due to the slight difference in heights between the two BPBs. Future research with more severe pulses is needed to better understand reclined children’s motion in far-side lateral-oblique impacts.

The influence of L-proline and fulvic acid on oxidative stress and semen quality of buffalo bull semen following cryopreservation.

This study investigates the effects of cryopreservation and supplementation of Azeri water buffalo's semen with proline (Lp) and fulvic acid (FA). Therefore, this study aimed to assess motility parameters, sperm viability, oxidative stress parameters, and DNA damage to detect the optimum concentrations of Lp and FA for buffalo semen cryopreservation. Thirty semen samples of three buffalo bulls were diluted in Tris-egg yolk extender and divided into 12 equal groups including control (C), Lp-10, Lp-20, Lp-40, Lp-60, Lp-80 (containing 10, 20, 40, 60, 80mM L-proline, respectively), FA-0.2, FA-0.5, FA-0.8, FA-1.1, FA-1.4 and FA-1.7 (containing 0.2%, 0.5%, 0.8%, 1.1%, 1.4% and 1.7% fulvic acid, respectively). The velocity parameters, TM and PM were improved by FA-1.7, FA-1.4, Lp-40 and Lp-60 groups compared to the C group but no significant difference was found regarding the amplitude of lateral head displacement and straightness compared to the control groups. The percentage of sperm viability and PMF were increased by FA-1.7, FA-1.4, FA-1.1, Lp-40 and Lp-60 groups compared to C group, while in terms of sperm DNA damage FA-1.7, FA-1.4, FA-1.1, Lp-10, Lp-20, Lp-40 and Lp-60 groups showed better results compared to C group. The results also showed that FA-1.7, FA-1.4, FA-1.1, Lp-20, Lp-40 and Lp-60 groups could improve TAC, SOD, GSH and decrease MDA levels. Also, FA-1.7, FA-1.4, Lp-20 and Lp-40 groups could improve GPx levels but just FA-1.7, and Lp-40 groups could improve CAT levels compared to C group. Thus, it can be concluded that L-proline and fulvic acid supplementations can improve the quality parameters of post-thawed buffalo bull semen.

Machine Learning Approach for Muscovy Duck (Cairina moschata) Semen Quality Assessment.

This study aimed to develop a comprehensive approach for assessing fresh ejaculate from Muscovy duck (Cairina moschata) drakes to fulfil the requirements of artificial insemination in farm practices. The approach combines sperm kinetics (CASA) with non-kinetic parameters, such as vitality, enzyme activities (alkaline phosphatase (AP), creatine kinase (CK), lactate dehydrogenase (LDH), and γ-glutamyl-transferase (GGT)), and total DNA methylation as training features for a set of machine learning (ML) models designed to enhance the predictive capacity of sperm parameters. Samples were classified based on their progressive motility and DNA methylation features, exhibiting significant differences in total and progressive motility, curvilinear velocity (VCL), velocity of the average path (VAP), linear velocity (VSL), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and live normal sperm cells in favour of fast motility ones. Additionally, there were significant differences in enzyme activities for AP and CK, with correlations to LDH and GGT levels. Although motility showed no correlation with total DNA methylation, ALH, wobble of the curvilinear trajectory (WOB), and VCL were significantly different in the newly introduced classification for "suggested good quality", where both motility and methylation were high. The performance differences observed while training various ML classifiers using different feature subsets highlight the importance of DNA methylation for achieving more accurate sample quality classification, even though there is no correlation between motility and DNA methylation. The parameters ALH, VCL, triton extracted LDH, and VAP were top-ranking for "suggested good quality" predictions by the neural network and gradient boosting models. In conclusion, integrating non-kinetic parameters into machine-learning-based sample classification offers a promising approach for selecting kinetically and morphologically superior duck sperm samples that might otherwise be hindered by a predominance of lowly methylated cells.