Eosin-nigrosin Staining Research Articles

PSVI-18 Association between residual feed intake and reproductive-related traits in young bulls

Abstract Enhancement of feed efficiency (FE) along with reproductive performance is critical to maximize profitability of beef production systems. Residual feed intake (RFI) is a measure of FE, assessing differences between expected and actual feed intake. Published data suggest a negative impact of RFI on reproductive performance. The objective of this experiment was to establish the relationship between RFI and reproductive parameters related with puberty in young bulls. Growing, Brangus bulls (n = 58; 10 to 12 mo of age) were tested for FE over 56 d and ranked as low RFI (LRFI) and high RFI (HRFI). Scrotal circumference (SC) was measured at the greatest diameter of the testicles by the same operator using a scrotal measuring tape, and semen samples were collected by electroejaculation at the beginning and at the end of the test. Fresh semen was visually evaluated (VE) for visual motility (VM), morphology [major defects (MAD)], minor defects (MID), total defects (TD)], plasma membrane integrity (PMI) via Eosin-nigrosin stain and sperm concentration (Con). Sperm kinematics were objectively determined simultaneously via computer-assisted sperm analysis (CASA-IVOS II System, Hamilton Thorne, Beverly, MA) within 6 h of collection in a medium designed specifically for holding sperm. Statistical analysis included repeated measures to assess interactions between Group (LRFI, HRFI) and Time (0 and 56 d of RFI measurement). Results are presented as mean ± SEM in Table 1. The main effect of Group did not affect CASA derived traits, while MAD and TD were affected among VE traits. A Time effect was observed in the amplitude of lateral head displacement (ALH), beat cross frequency (BCF), curvilinear velocity (VCL) and linearity (LIN). A tendency was indicated for total motility (TM), and average path velocity (VAP) among CASA derived traits, while all VE traits were affected by Time except for visual motility (VM). Finally, an interaction effect (Group x Time) was observed in LIN and plasma membrane integrity (PMI). In conclusion, age at puberty as determined by VM, Con, and SC (Group: P = 0.12, Day: P < 0.001, Group x Day: P = 0.79) was not associated with RFI. Overall, fertility-related measurements were not influenced by RFI, with time emerging as the primary influencing factor across evaluated parameters. Evaluation of reproductive parameters following d 56 FE testing may be a useful determinant for identifying differences in seminal characteristics of young bulls.

Comparison of Special Stains for Analysing the Morphology of Sperm: A Cross-sectional Study

Introduction: In India, male infertility constitutes 40% of infertility cases in males, which has become a serious problem in developing countries. Numerous studies and advanced techniques have been identified in assessing sperm morphology. The detection of sperm morphology is a simple and widely accepted method for determining sperm viability. It assists in selecting appropriate treatments for male infertility in assisted reproductive techniques. Infertility issues can arise in healthy males due to altered sperm morphology. Aim: To analyse the morphology of sperm using basic histopathology stains such as Haematoxylin and Eosin (H&E), Giemsa (G), Eosin-Nigrosin (EN), and Papanicolaou (Pap) stains. Materials and Methods: A cross-sectional study was conducted from April 2022 to June 2022 at the Department of Clinical Pathology, Sree Balaji Medical College and Hospital, Chennai, Tamil Nadu, India. Thirty semen samples were collected from healthy males and stained with H&E, Giemsa, Eosin-Nigrosin, and Papanicolaou stains. The results were observed and tabulated based on sperm shape, size, and motility using Kruger’s strict morphology method. A comparison was made to identify the fastest and most cost-effective method among the four stains. Descriptive statistics were used for the comparison. Results: Among the 30 semen samples, H&E and Papanicolaou stain methods were found to be rapid and cost-effective for analysing sperm morphology compared to the other stains (p-value <0.001). The findings included cases of Normozoospermia (18), Oligozoospermia (9), Necrozoospermia (1), and Teratozoospermia (2). In the Papanicolaou stained samples, the mean sperm head length (μm) was 8.92a±0.5, head width (μm) was 4.48a±0.33, head perimeter (μm) was 27.69a±1.85, head area (μm²) was 33.96a±3.74, tail length (μm) was 44.31a±2.02, sperm length (μm) was 53.24a±2.18, with no cytoplasmic droplets greater than half the size of the head and two vacuoles observed. Papanicolaou stain was the least expensive stain (9.50rs) compared to the other stains used for assessing sperm morphology. Conclusion: Papanicolaou stain is a simple and cost-effective method for analysing sperm morphology compared to other special stains. However, EN Stain is used to detect the morphology of live sperm. Sperm morphology assessment is necessary to monitor the causes of male infertility and determine appropriate treatment modalities.

Reproductive characteristics of Saanen and Alpine bucks

Genetic variations among breeds within a species can impact not only productivity traits, such as milk yield and quality, but also animal health, including fertility. This study aimed to compare the reproductive characteristics of bucks from the Saanen and Alpine breeds. Sperm concentration and motility were assessed using light microscopy, viability was determined using eosin-nigrosin staining, and morphological parameters were evaluated using the Spermac Stain method. DNA fragmentation was measured using the Halosperm kit. Artificial insemination of goats was conducted with fresh semen during natural estrus. Statistical analysis was performed using the Graph Pad Prism software. The results revealed that Alpine bucks exhibited significantly higher semen volume, sperm concentration, viability, and motility (P<0.05). No significant differences (P≥0,05) were observed between the breeds regarding the number of spermatozoa with normal morphology and the rate of DNA fragmentation. Cryobiological analysis of spermatozoa from Saanen bucks suggested a higher cryoresistance compared to the Alpine breed. Following artificial insemination of goats, the pregnancy rate for the Saanen breed was 61.8%, which was twice as high as that observed in Alpine goats — 28.8% (P<0.05). These findings demonstrate significant differences in reproductive characteristics between Saanen and Alpine goats. Despite superior sperm characteristics, the pregnancy rate after artificial insemination was significantly lower in the Alpine breed compared to the Saanen breed. Consequently, it is crucial to consider these variations in essential reproductive characteristics when implementing breeding programs and employing reproductive biotechnology in animal husbandry to ensure their successful application and effectiveness.

Eosin Nigrosin staining technique in assessment of sperm vitality in medical laboratories – A snippet from our experience on implementing the staining, interpretation and quality control procedures

Eosin Nigrosin staining for assessment of sperm vitality is an essential component of basic semen analysis as it helps differentiate between dead and immotile sperms, and has clinical implications in terms of patient treatment and follow up. This staining technique involves minimal use of reagents and simple procedural steps. Standardization of the same is pertinent to warrant accurate and reproducible results in medical laboratories, even those not specialized in infertility care. We wish to share our hands on experience through various stages in implementing this staining technique from challenges faced to putting quality control processes in place as reference for peers.

A matter of agreement: The effect of the technique and evaluator on the analysis of morphologic defects in stallion sperm.

Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High: >57% normal sperm, Moderate: 23-56% normal sperm, or Low: <23% normal sperm (four stallions per category). The data were analyzed using three different statistical methods: Analysis of Variance (ANOVA), correlative analysis, and Bland-Altman method (agreement). A higher level of agreement among techniques was observed between DIC and PH for morphologically normal sperm. The agreement between the alternative methods (EN, DQ, or PH) and the standard method (DIC) varied, depending on the sperm quality level (High, Moderate, or Low). Some morphological defects (e.g., AH, AMP) were constantly underestimated with the staining methods (DQ, EN) compared to DIC and PH, particularly in ejaculates with low sperm morphology. Underestimation of some abnormalities, due to the technique or the evaluator, has the potential to alter the clinical interpretation of stallion fertility.

Defining an Optimal Range of Centrifugation Parameters for Canine Semen Processing.

Our objective was to determine a clinically relevant range of centrifugation parameters for processing canine semen. We hypothesized that higher gravitational (g) force and longer time of centrifugation would result in improved spermatozoa recovery rate (RR) but poorer semen quality. Cooled storage under standard shipping conditions was used as a stressor to evaluate long-term treatment effects. Individual ejaculates collected from 14 healthy dogs were split into six treatment groups (400 g, 720 g, and 900 g for 5 or 10 min). Sperm RR (%) was calculated post-centrifugation, and plasma membrane integrity (%, Nucleocounter® SP-100™), total and progressive motility (%, subjective and computer-assisted sperm analysis), and morphology (%, eosin-nigrosin staining) were assessed on initial raw semen (T0), post-centrifugation (T1), and 24 h (T2) and 48 h (T3) after cooling. Sperm losses were minimal, and RRs were similar across treatment groups (median >98%, p ≥ 0.062). Spermatozoa membrane integrity was not different between centrifugation groups at any time point (p ≥ 0.38) but declined significantly during cooling (T1 vs. T2/T3, p ≤ 0.001). Similarly, total and progressive motility did not differ across treatments but declined in all groups from T1 to T3 (p ≤ 0.02). In conclusion, our study showed that centrifugation within a range of 400 g-900 g for 5-10 min is appropriate for processing canine semen.

Evaluation of Motility, Viability and Abnormality of Boar Spermatozoa in Various Modified Extenders

The purpose of this study was to conduct an assessment on the quality of pig semen spermatozoa during storage and determine which diluent material should be used and how long pig semen can be stored. Freshly ejaculated boar semen was obtained from a two-years-old Duroc male and was diluted with Beltsville Thawing Solution (BTS) and Tris Citrate Fructose (TCF) supplemented with 5% egg yolk (KT) and extract of Borassus flabellifer Linn mesocarp (EM) 0.01% and stored at 13°C for up to 4 days. An experimental study with a completely randomized design with four groups and five repeats was applied. Parameters observed were motility, viability and abnormality of spermatozoa. Sperm quality was evaluated by examining motility and abnormality using a phase-contrast microscope with a magnification of 100X, and viability by eosin nigrosine staining. Parametric data were analyzed with ANOVA followed by Duncan test. The results showed that the supplementation of 5% egg yolk in the BTS diluent-based treatment group showed a higher percentage of motility (P&lt;0.05) than without egg yolk, while the TCF-based diluent group showed no differences (P&lt;0.05). Modified-Tris Citrate Fructose diluent supplemented with 5% egg yolk and 0.01% mesocarp extract was able to maintain a significantly higher percentage of viability (67.50±2.0) (P&lt;0.05) than other groups, and had a percentage of abnormalities (9.75±0.957), significantly lower (P&lt;0.05) than other groups. It was concluded that the modification of diluent TCF with 5% egg yolk and mesocarp extract 0.01% provide the best results in maintaining sperm quality with the highest percentage of sperm motility and viability, and the lowest percentage of sperm abnormality compared to the other three diluents. Likewise, all groups of modified-BTS and TCF diluent supplemented with 5% egg yolk and extract of Borassus flabellifer Linn mesocarp in this study were able to maintain motility &gt;40%, viability &gt;45% and abnormalities &lt;20% so they were appropriate for using in the AI procedure.

In vitro effects of plasma rich in growth factors on human teratozoospermic semen samples

There is a correlation between teratozoospermia and production of reactive oxygen species leading to poor assisted reproductive techniques outcomes. This study aimed to examine the effect of plasma-rich in growth factors (PRGF) on teratozoospermic samples. Twenty-five teratozoospermic samples were included in this study. After sperm preparation, it was divided into four groups, including 0 (control), 1, 5, and 10% PRGF. Sperm motility, viability (eosin-nigrosin staining), morphology (Papanicolaou staining), DNA fragmentation (sperm chromatin dispersion test), mitochondrial membrane potential (JC-1 staining by flow cytometry), and lipid peroxidation (measurement of malondialdehyde, MDA) were evaluated before and after 1 h of incubation with or without PRGF. Our results showed that after 1 h of incubation, the addition of 1% PRGF improved sperm progressive motility (47.72 ± 13.76%) compared to the control group (17.36 ± 8.50%) (p < 0.001). Also, 1% PRGF preserved the sperm’s total motility (77.50 ± 13.28% vs. 65.63 ± 19.03%, for 1% PRGF and control, respectively) and viability after incubation. The rate of normal sperm morphology was the same between different groups. Higher mitochondrial membrane potential and lower DNA fragmentation were also observed in sperm treated with different concentrations of PRGF compared to the control group, but the differences were non-significant. The MDA levels were significantly decreased in PRGF-treated groups compared to the control group (0.99 ± 0.62, 0.95 ± 0.33, 0.95 ± 0.79, and 1.49 ± 0.27 for 1% PRGF, 5% PRGF, 10% PRGF and control, respectively). Based on our results, it seems that PRGF incubation can improve sperm parameters and especially decrease the level of malondialdehyde as an indicator of oxidative stress, which is one of the main problems of teratozoospermic samples.

Influence of trans-ferulic acid on the quality of ram semen upon cold preservation.

Due to lower antioxidant capacity and higher amounts of polyunsaturated fatty acids, ram spermatozoa are very susceptible during cooling process. The objective was to examine the effect of the trans-ferulic acid (t-FA) on the ram semen during liquid preservation. Semen samples were collected from the Qezel rams, pooled, and extended with the Tris-based diluent. Pooled samples enriched with different amounts of the t-FA (0, 2.5, 5, 10, and 25mM) and preserved at 4°C for 72h. Spermatozoa's kinematics, membrane functionality, and viability were assessed by CASA system, hypoosmotic swelling test, and eosin-nigrosin staining, respectively. Moreover, biochemical parameters were measured at 0, 24, 48, and 72h. Results showed that 5 and 10mM t-FA improved forward progressive motility (FPM) and curvilinear velocity compared to the other groups at 72h (p < 0.05). Samples treated with 25mM t-FA showed the lowest total motility, FPM, and viability at 24, 48, and 72h of storage (p < 0.05). Higher total antioxidant activity levels were observed in the 10mM t-FA-treated group compared to the negative control at 72h (p < 0.05). Treatment with 25mM t-FA increased malondialdehyde amounts and decreased superoxide dismutase activity compared to other groups at the final time assessment (p < 0.05). Nitrate-nitrite and lipid hydroperoxides values were not affected by treatment. The current study indicates the positive and negative influences of different concentrations of t-FA on the ram semen upon cold storage.

The Quality of Frozen Friesian Holstein Semen after Long-Term Storage

Semen cryopreservation is the long-term storage at very low temperatures in liquid nitrogen for future use. This study investigates the quality of frozen Friesian Holstein (FH) semen after long-term storage. Samples of FH semen stored for 25, 20, 15, 10, 5, and 1 years were collected from one of the national centers for artificial inseminations. Frozen semen was stored in containers with liquid nitrogen at -196 ᵒC in a room with a temperature of 20 ᵒC The variables used after thawing were sperm motility, viability, and abnormalities, as well as plasma membrane integrity (IPM) using computer-assisted sperm analysis (CASA), eosin-nigrosine staining, and hypoosmotic swelling (HOS) test, respectively. Data were analyzed by one-way ANOVA and expressed as mean ± SEM. The result showed no significant differences in sperm viability, abnormalities, and IPM. Furthermore, sperm motility was >40%, consistent with the Indonesian standard for frozen bovine semen. CASA analysis showed that all variables of the motility pattern have no significant difference, except linearity (LIN). The Lin of sperm was lower in frozen semen after one and five years than after 20 and 25 years of storage. The overall quality of semen after 25, 20, 15, 10, 5, and 1 years of storage met the standard and was suitable for artificial insemination.

Donkey semen cryopreservation: Alternatives with permeable, non-permeable cryoprotectants and seminal plasma.

Cryopreservation of semen is an important technique to preserve genetic material. Yet, pregnancy rates in jennies after artificial insemination with frozen-thawed donkey semen are poor. This condition has been attributed to the impact of permeable cryoprotectants, that could cause high post-breeding endometritis. Removal of seminal plasma (SP) prior to semen freezing process is another contributing factor. SP is involved in a multitude of sperm functions and events preceding fertilization and has a mediating effect of sperm capacitation and postcoital uterine inflammatory response. The aim of this study was to evaluate different alternatives in donkey semen cryopreservation with permeable, non-permeable cryoprotectants, BSA and SP. Thirty ejaculates from 10 donkeys were cryopreserved with different combinations of dimethylformamide (DMF, 5%), sucrose (SUC, 200 mM) and homologous SP (10%): DMF (T1), DMF/SP (T2), SUC/BSA (T3), SUC/BSA/SP (T4), DMF/SUC/BSA (T5), DMF/SUC/BSA/SP (T6), DMF/BSA (T7) and DMF/BSA/SP (T8). After thawing, sperm motility and kinetics were assessed by computerized semen analysis. Sperm vitality (SV) was evaluated by fluorescence microscopy, functional membrane integrity (FMI) by the HOST test, abnormal morphology by eosin-nigrosin staining and sperm membrane stability by flow cytometry. For statistical analysis, sperm quality indexes (SQi) were obtained, general linear models were carried out and mean comparisons were made by the Tukey test. T1, T2, T5, T6, and T7 had higher and equivalent results for motility, most kinetic parameters and function membrane integrity. Cryopreservation of donkey semen without permeable cryoprotectant (T3 and T4) showed a reduction in motility, kinetics, SV, FMI and SQi. T5 showed a reduction in progressive motility, sperm velocities, IMF and SQi compared to other DMF treatments. T6 and T8 achieved higher SQi values compared to T1, but they were not different compared to T2 and T7. T1 had a smaller sperm population with low-M540 compared to T3. It is concluded that the use of permeable cryoprotectant is essential to achieve higher post-thaw quality of donkey semen. In addition, the combined use of BSA, SUC and/or PS may provide additional sperm protection compared to the individual use of DMF.

Relação da qualidade do sêmen com endogamia e marcha de cavalos crioulos colombianos

ABSTRACT: High consanguinity among equines has negative effects on semen quality, thus resulting in low motility and high levels of abnormality in the spermatozoa. However, such a relationship has not been studied in Colombian Creole horses, which have been subjected to particular selection practices focusing mainly on their gait. This research assessed the relationship of semen quality to inbreeding and gait of Colombian Creole horses. Semen was collected from 50 horses using the artificial vagina method. Sperm motility and kinematics were assessed with a computerized analysis system (SCA®). Sperm vitality (SV) and abnormal morphology (AM) were assessed via the eosin-nigrosin staining test. Functional membrane integrity (FMI) was assessed via the hypo-osmotic swelling test (HOST). Genealogies and consanguinity analysis was conducted using the Breeders Assistant for Horses program. An average of 3.6 ± 0.4 % was reported for the inbreeding coefficient (Ft). A decrease in sperm motility and kinematics was reported, which was associated with an increase in consanguinity (P &lt; 0.05). Furthermore, differences in consanguinity were found based on gait. Similarly, a relationship between horse gait and semen quality (P &lt; 0.05) was found. Authors concluded that semen quality of Colombian Creole horses has been affected by inbreeding and its relationship with genetic selection based on gait.

Sperm Membrane and Acrosomal Integrity Affected by using Black Currant

This study was designed in order to investigate and determine the effect of black current (Prunus cerasus) extract on semen membrane and acrosome integrity of the rams. Twelve mature healthy rams were used in this study aged between 2-3 years, kept in the animal farm of the College of Veterinary Medicine, Al-Kufa University. Semen ejaculates was collected using electro-ejaculator methods once weekly a total of 36 ejaculates were collected among the period of the study. The black current (Prunus cerasus) extract was prepared by using maceration and added to the diluted semen in five doses groups (0.25 gm/ml as G1, 0.5 gm/ml as G2, 1 gm/ml as G3, 2 gm/ml as G4, 4 gm/ml as G5) and the positive control group left without any addition. The treatment samples were evaluated under cooling for five consecutive days, the membrane integrity test was evaluated using hypo-osmotic solution and acrosome integrity was analysis using semen smear stained with eosin–nigrosin stain. The result showed that high concentration of aqueous plant extract give negative result as listed in G3, G4 and G5 in the all preservation days. As found in acrosome integrity at the 5thday of incubation showed decreased at highly significantly sequentially. We conclude that the G1 and G2 of black current extract (Prunus cerasus) should be used in chilling process as a part of artificial insemination in sheep due to its protective effect of the membrane and acrosome integrity.

Post-Thaw Characteristics of the Simmental Sperm Function in Different Ages of Bulls

This study aims to determine the effect of the age difference of Simmental bulls on motion characteristics, capacitation status, and DNA fragmentation of post-thawing sperms. The frozen semen used was collected from twelve bulls, which were divided into four groups of age, which include a group of two, four, ≥ 10 years old with high semen rejection (≥ 10 HR), and ≥ 10 years old with low semen rejection (≥ 10 LR). Computer Assisted Sperm Analysis (CASA) was used to determine sperm motion characteristics, capacitation status by chlortetracycline (CTC) staining, plasma membrane integrity, and viability using eosin-nigrosine staining. In contrast, the DNA fragmentation index was determined using the Sperm-Bos-halomax kit. The results showed that the four year old group had a higher total and progressive motility percentage than the others (p<0.05). In all groups, there was no significant difference among sperm kinematics such as VAP, VSL, VCL, STR, and ALH. However, the LIN, WOB, and BCF of the ≥ 10 HR year old groups were significantly lower (p<0.05) than those of the other groups. However, un-capacitated sperm was higher (p<0.05) in the two years and four years old groups compared to the ≥ 10 years old, while the four years old group had lower capacitated and acrosome-reacted (p<0.05) than the other groups. Furthermore, the sperm membrane integrity, viability, and DNA fragmentation index of the ≥ 10 years old groups were significantly higher (p<0.05) than those of the other groups. The research concludes that aging in the Simmental bull affects motion characteristics, capacitation status, and DNA fragmentation of post-thawing sperm. However, the semen rejection rate in the older bull did not directly affect the post-thawing sperm quality.

Semen characterization of Dang Surat Thai native chicken

Native Dang Surat chicken play an important role in poultry breeding of southern Thailand but their production performance falls short of commercial counterparts. Nevertheless, native birds have superior disease resistance while meat texture and taste of eggs are popular among Thais. Husbandry of native breeds are confined to smallholders who can only fulfil local niche outlets. To satisfy the wide-spread demand throughout Thailand and further afield, local-breed sires are crossed with high productivity hens. To maintain a constant supply of F1 hybrids, a colony of Dang Surat cocks are maintained by the Department of Livestock who select by phenotypes, not semen quality that determines male productivity. Accordingly, semen volume, pH, color and consistency, sperm motility, concentration, viability were measured. Morphology was also assessed by using eosin-nigrosin staining. Semen was slightly alkaline (pH, 8.0±0.1), mean volume=265±23 μL, had high sperm concentrations, 2740±76 x106/ml, of which 98.9±0.1% were viable. Abnormal sperm heads, tails and mid pieces, cytoplasmic droplets and detached heads comprised 1.5±0.3, 3.7±0.4, 0.3±0.1 and 0.8±0.1%, respectively. The findings showed normal seminal fluids and sperm parameters similar to commercial poultry semen in general. Our data will help develop efficient cockerel selection, natural flock breeding plan, and assisted reproduction by cryopreservation and artificial insemination to better meet market demands.

Liquid storage of Ostrich (Struthio camelus) semen at 5 °C through intermediate dilution

Dilution rate, dilution temperature and storage time have been recognized as vital steps in the processing of semen for storage before artificial insemination. The objective of this study was to determine optimal dilution and dilution temperature with an ostrich-specific semen extender for chilled storage. Four preselected ostrich (Struthio camelus var. domesticus) males, known for their ease of collection and specific semen quality parameters, were collected using the “dummy” female method. Dilution of 384 semen samples, at rates of 1:1, 1:2, 1:4 and 1:8 semen/diluent ratio with a diluent set at 5, 21 and 38 °C was performed and stored for 48 h at 5 °C. In vitro sperm function tests were conducted to evaluate treated semen during different storage intervals of 1, 5, 24 and 48 h. Motility and kinematic parameters were measured by the Sperm Class Analyzer®, the percentage live sperm measured by fluorescence SYBR14®/PI (LIVE/DEAD®), the percentage of sperm able to resist the hypo-osmotic swelling (HOS) stress test and sperm morphology determined by Nigrosin-Eosin staining. Progressive motility (PMOT), motility (MOT), sperm kinematics, LIVE and HOS were best (P < 0.05) maintained at a higher dilution of 1:4–1:8. The beneficial effect (P < 0.05) of a higher dilution temperature (21 °C) was prominent in terms of PMOT at a higher dilution. Storage of chilled semen at 5 °C requires dilution, at interpolated rates of 1:6–1:7, together with an extender temperature of 21 °C, to maintain optimal sperm function with minimal loss over a 48 h storage period.

Sodium dodecyl sulphate, N-octyl β-D glucopyranoside and 4-methoxy phenyl β-D glucopyranoside effect on post-thaw sperm motion and viability traits of Murrah buffalo (Bubalus bubalis) bulls

Sodium Dodecyl Sulphate (SDS), N-Octyl β-D Glucopyranoside (NOG), 4-Methoxy Phenyl β-D Glucopyranoside (4-MPG) as ice recrystallization inhibitors were added to Tris Egg Yolk Glycerol (TEYG) semen extender for cryopreservation of semen of buffalo bulls. Post-thaw sperm motion and viability traits were evaluated. Pilot study involved six semen ejaculates (2 ejaculates/bull, from three bulls); second experiment was conducted using twenty seven semen ejaculates (9 ejaculates/bull, from 3 bulls) and in third experiment three semen ejaculates (one bull) were used. Eight concentrations of SDS (2, 1, 0.5, 0.25, 0.15, 0.125, 0.0625 and 0.0312%), twelve concentrations of NOG (33, 22, 11, 5.5, 2.5, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.0156 mM), and, eleven concentrations of 4-MPG (220, 165, 110, 55, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mM) were supplemented in TEYG semen extender to evaluate the post-thaw sperm motility and viability traits. Computer Assisted Sperm Analysis (CASA) was used to measure the kinetic and functional parameters for sperm motion traits, Hypo Osmotic Swelling Test (HOST) for sperm plasma membrane integrity, Eosin Nigrosin staining for viability and Rose Bengal staining for sperm abnormalities for all the experiments except for pilot study where only Total Motility (TM) and Rapid Progressive Motility (RP) were evaluated. Three freezing protocols; i) Normal P24 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −40 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); ii) Moderate P25 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −50 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C); and iii) Rapid P26 (freezing rate of −30 °C min−1 from 4 °C to −15 °C; −60 °C min−1 from −15 °C to −60 °C; and −50 °C min−1 from −60 °C to −140 °C; and then plunged in liquid Nitrogen at −196 °C) were evaluated using SDS 0.125% in TEYG semen extender. SDS ≤0.125%, NOG ≤0.0625 mM and 4-MPG ≤ 3.125 mM in TEYG buffalo semen extender improved significantly (p < .05) the kinetic and functional parameters as compared to the other Ice Recrystallization Inhibitors (IRIs) concentrations used for cryopreservation of buffalo bull semen in the pilot study. SDS 0.125% supplementation was the best IRI among all which resulted in improved kinetic and functional parameters of bull semen in second experiment. Conclusion was drawn that buffalo bull semen cryopreservation using sodium dodecyl sulphate, 0.125% as IRI in TEYG semen extender along with freezing protocol P 25 revealed optimum kinetic and functional parameters for post-thaw spermatozoa.

A retrospective study of the relationship between canine age, semen quality, chilled semen transit time and season and whelping rate and litter size

The objectives of this study were to investigate the effect of the dog’s age, semen quality, and the duration and season of semen transit on whelping rate and litter size after insemination with transported chilled extended semen. The sperm rich fraction was collected from 43 dogs of 18 breeds, which were presented at the Clinic for chilled semen transport, in the period from 2017 to 2021. Immediately after collection, the total sperm concentration and count, motility, membrane integrity (HOS test), the percentage of live spermatozoa and sperm morphology (eosin nigrosin staining) were evaluated. The sperm rich fraction was centrifuged and diluted with Tris – fructose - citrate extender with the addition of 20% (v/v) egg yolk, then chilled and prepared for shipping. A dose consisted of at least 200x106 live, motile, morphologically normal spermatozoa. The data on the dog’s age, chilled sperm transit time, the season of transit, and the whelping rate and litter size after insemination were recorded. The whelping rate was 55.8% with a mean (±SEM) litter size of 4.71±0.58 pups. The total number of spermatozoa was higher in artificial insemination (AI) that resulted in whelping compared to unsuccessful AI (P&lt;0.05). No difference was observed for any of other sperm quality parameters tested, such as the dog’s age or season of transit regarding whelping rate or litter size. Transit time significantly affected the whelping rate (P&lt;0.01), at (mean±SEM) 21.50±1.28 and 37.00±5.59 h in successful and unsuccessful AI, respectively. In conclusion, analysis of the factors related to the dogs identified total sperm count and transit time as factors that significantly affected whelping rates in bitches inseminated with transported chilled extended semen.

P-056 The reproductive toxicity of alginate coated silver nano-rod on sperm

Abstract Study question What is the toxic effects of Au@Ag core-shell NR on sperm function? Summary answer Although sperm count and morphology did not differ significantly, changing the shape and envelope of the silver nanostructure did not reduce its toxicity. What is known already Spermatogenesis is a developmental process of germ cell reproduction that depends on hormonal or dynamic interplays between sertoli cells and germ cells. Silver is the most common noble metal used in the synthesis of nanoscale materials, particularly in room sprays, cosmetics, toys, clinical catheters, and wound dressings. The increasing usability of silver nanoparticles (Ag NP) has increased human exposure. Many studies have demonstrated that Ag nanoparticles, depending on their shape, size, and concentration, have cytotoxic effects that reduce cell viability. The toxicity of silver Nano-Rod by deformation and coating with alginate has been studied. Study design, size, duration Forty-eight male mice were divided into five groups (two treatments, two shams, and one control). The treated groups received the Ag Nano-Rod at a dose of 1.5 mg/kg/day intraperitoneally for fourteen and thirty-five days. The sham groups were treated with identical amounts and days, and the control group received no material. Finally, the cauda epididymis of the animals was removed. Participants/materials, setting, methods In the present study, Ag Nano-Rod were characterized by HR-TEM, FT-IR, UV-visible, XRD and ICP-MS. Sperm analysis, including count, motility, viability, and morphology, was studied according to the criteria of WHO. A phase-contrast microscope was used to count the spermatozoa and assess their motility. Viability and morphology were examined by eosin-nigrosin and papanicolaou staining, respectively. TUNEL assay and gene expression for apoptotic and autophagic markers were also performed. Main results and the role of chance High magnification images TEM were obtained. According to this, the Ag nanostructure was rod-shaped and the average size of the particles was 50nm. The UV-visible confirms the formation of the synthesized nanosilver (rod shape). The change in nanostructure coating from CTAB to alginate was studied by FTIR analysis, and XRD proved the presence of silver in the composition. The results of sperm analysis showed that there was no significant decrease in sperm count and morphology but the parameters of motility and viability of the nanosilver groups showed a significant decline compared to the control group (P &amp;lt; 0.05). Assessment of sperm DNA fragmentation by the TUNEL assay showed that the nanosilver groups had significantly higher DNA fragmentation and the real-time PCR results also confirmed this. Expression of autophagic and apoptotic markers indicates activation of the apoptosis pathway compared to the autophagy pathway. The expression of LC3 and Beclin-1 (markers that initiate and continue the autophagy) was reduced compared to the sham and control groups. Also, examination of apoptotic pathway genes showed a decrease in Bcl-2 expression and an increase in Bax and caspase-3 expression. Limitations, reasons for caution This study requires further research, including protein expression in these two pathways. Wider implications of the findings This study investigated the acute (14 days) and chronic (35 days) effects of silver nanorod on reproductive toxicity, which can lead to significant changes in sperm quality. This reduction in quality was observed mainly in the chronic phase and severely affected sperm motility. Trial registration number Not Applicable

Heterospermično osjemenjivanje u pasa

The idea of dual sire insemination in dog breeding is to give equal chances to both males to fertilize the eggs due to the specific oestrus cycle of the bitch. Two sexually mature, privately owned females Petit Basset Griffon Vendeen (bitch A and B) aged 3 to 5 years were subjected to dual-sire inseminations. Prior to insemination, the semen of stud dogs (n=5) was collected and evaluated for volume, concentration, total sperm count, motility, membrane integrity (HOS test), percentage of live spermatozoa and sperm morphology (eosin nigrosin staining). The time of insemination was based on serum progesterone (P4) concentrations, where P4 of &amp;gt;5–10 ng/mL was considered ovulation. Bitch A was inseminated in two oestrus cycles with fresh mixed semen from two males using the endoscopic transcervical insemination technique (TCI). Bitch B was inseminated in one oestrus cycle by laparoscopic intrauterine deposition of frozen thawed semen. Semen evaluation showed minimum deviations in all tested parameters between the chosen males on the days of insemination. Depending on sperm concentration and quality, the volume of ejaculates was adjusted, resulting in an equal number of motile spermatozoa from two males in each insemination, providing them an equal chance for fertilisation. Confirmation of pregnancy was carried out 24 days after insemination by ultrasonography. The whelping outcome was obtained directly from the owner of the bitches. Puppy blood was taken immediately after whelping from the umbilical vein into EDTA tubes. Blood samples were also obtained from the dam and both sires for DNA profiling. Parentage was determined for each dual-sired pup by using Thermo Scientific Canine Genotypes Panel 1.1. All inseminations resulted in pregnancy and whelping. A total of 14 puppies were born in three litters. Mixed parentage was determined in 1 of the 3 resultant litters (bitch A). In conclusion, dual sire insemination is a useful breeding tool providing the opportunity to obtain puppies from multiple genetic backgrounds in a single litter. However, as this method usually produces offspring from single father, optimal insemination protocol should be established for producing a litter of mixed paternity.