Eosin-nigrosin Staining Research Articles

Sperm Vitality and Necrozoospermia: Diagnosis, Management, and Results of a Global Survey of Clinical Practice.

Sperm vitality testing is a basic semen examination that has been described in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen from its primary edition, 40 years ago. Several methods can be used to test sperm vitality, such as the eosin-nigrosin (E-N) stain or the hypoosmotic swelling (HOS) test. In the 6th (2021) edition of the WHO Laboratory Manual, sperm vitality assessment is mainly recommended if the total motility is less than 40%. Hence, a motile spermatozoon is considered alive, however, in certain conditions an immotile spermatozoon can also be alive. Therefore, the differentiation between asthenozoospermia (pathological decrease in sperm motility) and necrozoospermia (pathological decrease in sperm vitality) is important in directing further investigation and management of infertile patients. The causes leading to necrozoospermia are diverse and can either be local or general, testicular or extra-testicular. The andrological management of necrozoospermia depends on its etiology. However, there is no standardized treatment available presently and practice varies among clinicians. In this study, we report the results of a global survey to understand current practices regarding the physician order of sperm vitality tests as well as the management practices for necrozoospermia. Laboratory and clinical scenarios are presented to guide the reader in the management of necrozoospermia with the overall objective of establishing a benchmark ranging from the diagnosis of necrozoospermia by sperm vitality testing to its clinical management.

The Viability of Bali Cattle Epididymal Sperm with the Addition of Caffeine in Tris-Egg Yolk Diluents

The research aimed to examine the effect of caffeine's addition on diluents on Bali cattle epididymis' survival or sperm viability to get the right amount of caffeine. The epididymis collection was taken from the Slaughterhouse of Ternate City. Analysis of sperm vitality or viability was carried out in the Biotechnology Laboratory of Universitas Khairun. The methods in this research were by preparing egg yolk Tris solution treated with caffeine P0 (0 mg/ml), P2 (2 mg/ml), P4 (4 mg/ml), and P6 (6 mg/ml) with four replications, and then the epididymal sperm was inserted into each treatment. The epididymal sperm viability was calculated by evaluating the percentage of the number of living sperm. It compared to the dead sperm, which were observed microscopically by eosin-nigrosin staining. The data obtained were analyzed by one-way analysis of variance (ANOVA) to see if there were significant differences (P<0.05) or very real (P<0.01), the analysis were then continued with the Tukey-W-Procedure test with SPSS 18. The results show that administration of caffeine 2 mg/ml and 4 mg/ml (86.95% and 86.95%) have a significant effect on the sperm viability of epididymis compared with 0 mg/ml and 6 mg/ml (69.38% and 69.91%) treatments.

78 Evaluation of extended hair sheep ram semen stored as liquid at 5°C

Abstract This study was designed to evaluate the quality of extended hair sheep ram semen stored as a liquid at 5°C. St Croix White (STX; n = 6) and Dorper x STX (DRPX, n = 5) rams were collected weekly for 3 wk using estrus ewes fitted with an intravaginal collection vial. Semen was kept at 32°C during transport to the lab and during processing. Semen was evaluated for percent motility (MOT), viability (LIVE) using eosin-nigrosin stain and concentration using a hemocytometer counting chamber. Semen was extended to a final concentration of 250 x 106/mL in a one-step dilution with a skim UHT milk extender with 10% egg yolk by volume and packaged into 0.5 mL straws. Straws were stored in a styrofoam box in a refrigerator at 5°C for 96 h, or in an Equitainer® set up using the manufacturer’s instructions, for 24 h at which time they were transferred to the styrofoam box in the refrigerator for 72 h. Semen was evaluated for MOT and LIVE at -1, 0, 24, 48 72 and 96 h relative to cooling. Semen traits were analyzed using GLM of SAS for repeated measures with ram, time, breed and cooling method in the model. There was no difference (P > 0.10) in MOT or LIVE over time between breeds or cooling method. One STX ram did not produce any samples that survived extension; one STX and four DRPX rams produced at least one sample that did not survive extension and all were removed from analysis. The MOT decreased (P < 0.0001) from 81.7 ± 2.9 % at -1 h to 52.2 ± 2.9% at 96 h. The LIVE decreased (P < 0.0001) from 83.1 ± 3.6% at -1 h to 50.4 ± 3.6% at 96 h. These results show that ram semen stored as a liquid at 5°C can maintain motility and viability for 96 h. Further studies will be conducted to evaluate cooling of extended ram semen and fertility after artificial insemination.

Influence of Season and Liquid Storage at 16 °C on Beni Arouss Bucks' Semen Quality.

Simple SummaryGoat production plays an important economic and social role in Northern Moroccan farming. Beni Arouss is an autochthonous North Moroccan goat breed. The use of artificial insemination can largely contribute to optimised preservation and dissemination of valuable traits of this breed and lead to improved productivity. During liquid storage, the stability of semen good quality remains crucial in order to provide greater flexibility between the artificial insemination center and farms where the insemination of does needs to be performed. The study aims to assess the effect of storage and season of the year on fresh semen of Beni Arouss goats. Every month, ejaculates were collected from bucks and were extended and stored at 16 °C for 24 h. Semen motility, viability and normal morphology were assessed at 0, 4, 8 and 24 h after collection. As expected, these parameters showed a significant reduction within 24 h of storage and during all seasons. However, semen collected in summer maintained a better quality after 24 h of storage at 16 °C than semen collected during the other periods. Therefore, the storage ability of Beni Arouss bucks’ semen stored at 16 °C was higher during the summer.The study aimed at determining the effect of storage and season on fresh semen of Beni Arouss goats. Ejaculates were collected at monthly intervals from seven mature bucks and were extended at a final concentration of 800 × 106 spermatozoa. ml-1 and stored at 16 °C for 24 h. Semen motility, viability and normal morphology were assessed at 0, 4, 8 and 24 h after collection. Motility and normal morphology parameters were recorded using computer-assisted sperm analysis (CASA) and viability was analyzed using eosin–nigrosin staining. As expected, motility, viability and normal morphology parameters showed a significant reduction within 24 h of storage and during all seasons (p < 0.05). However, semen collected in summer maintained a better quality after 24 h of storage at 16 °C than semen collected during the other periods (p < 0.05). In conclusion, the storage ability of Beni Arouss bucks’ semen stored at 16 °C was significantly higher during the summer.

Effect of low-density lipoproteins and trehalose on the quality of cryopreserved bovine semen

Background: In artificial insemination, chicken egg yolk is added to bovine semen to protect it during the cryopreservation process, although it contains substances that can affect the microbiological quality and metabolism of sperm. Objective: To evaluate post-thaw quality of bovine cryopreserved semen added with centrifuged and non-centrifuged egg yolk, low-density lipoproteins (LDL), and trehalose (T). Methods: Ten ejaculates from five bulls were cryopreserved under the treatments T1: pure egg yolk (PEY) at 20% v/v, T2: centrifuged egg yolk (CEY) at 20% v/v, T3: LDL at 8% v/v, T4: T at 100 mM, and T5: T at 100 mM plus LDL at 8% v/v (TLDL). Spermatic motility and kinetics, functional membrane integrity (FMI), structural membrane integrity (SMI), sperm vitality (SV) and abnormal morphology (AM) were assessed using the Sperm Class Analyzer (SCA®) system, hypoosmotic test (HOST), SYBR/PI probes, and eosin–nigrosin staining, respectively. A completely randomized design was used. Normal distribution of the variables was validated through the Kolmogórov– Smirnov test. A generalized linear model was used to determine sources of variation. Means were compared using the Tukey test. Results: Inclusion of CEY or LDL had a similar effect on sperm protection, and were superior for motility, kinetics and membrane integrity compared to the other treatments (p&lt;0.05). CEY was superior for progressive motility (p&lt;0.05). The cryoprotective action of LDL was similar to TLDL for motility and kinetics, SMI, SV, and AM (p&lt;0.05). Inclusion of PEY and T resulted in the lowest semen quality (p&lt;0.05). The use of T resulted in a reduction in FMI and SMI (p&lt;0.05). No differences in AM between treatments were found (p&gt;0.05). Conclusions: Egg yolk can be replaced by centrifuged egg yolk or low-density lipoproteins in the freezing extender used for bovine semen used in artificial insemination.

Alpha-tocopherol improves sperm quality by regulate intracellular Ca2+ intensity (influx/efflux) of Simmental bull cattle sperm.

BackgroundThe effects of α-tocopherol on intracellular Ca2+ intensity in semen cryopreservation by regulate intracellular Ca2+ intensity have not been reported yet.ObjectiveThe research was conducted to evaluate the effect of supplementation α- tocopherol into egg yolk skim milk extender on sperm quality and intracellular Ca2+ intensity.MethodsSemen samples were collected and supplemented with respectively 0mM (P0); 0.5mM (P1); 1mM (P2); 1.5mM (P3) and 2mM (P4) α-tocopherol in extender before cryopreservation processes. Post-thawing sperm was evaluated for motility, viability, and abnormality using Phase Contrast Microscope (200x) with eosin-nigrosine staining, and intracellular Ca2+ intensity of the best result dose was evaluated using Confocal Laser Scan Microscope (400x) with Fluo-3 Staining.ResultsThe results showed there was a significant difference (P≤0.05) in sperm motility and viability between P0; P1 with P2; P3; P4. The Motility and viability between groups P0; P1 and P3; P4 showed no significant difference (P≥0.05), while P2 with P3; P4 showed significant difference (P≤0.05). There was a significant difference (P≤0.05) in sperm abnormality of P0; P1 with P2; P3; P4. The abnormality between P0; P1 and P2; P3 showed no significant difference (P≥0.05), while P2; P3 showed a significant difference with P4 (P≤0.05). The best result in sperm quality was supplementation with 1.5mM α-tocopherol. Ca2+ intracellular intensity: 142.76± 21.8 au (P0) and 176.06±61.43 au (P3).ConclusionsIt was concluded that 1.5mM α-tocopherol is the best dose to improve sperm quality by regulating intracellular Ca2+ intensity on Simmental bull cattle.

Soy lecithin as a potential alternative to powdered egg yolk for buck sperm cryopreservation does not protect them from mitochondrial damage

The aim of this study was to address whether soy lecithin (SL) was an effective non-penetrating cryoprotectant for buck sperm cryopreservation in the presence of seminal plasma. There was also an attempt to determine the optimal concentration of BHT as an antioxidant in powdered egg yolk (PEY) or in SL based media. Two ejaculates were collected from six bucks and mixed ejaculates were aliquoted into washed, using centrifugation procedures, and unwashed samples. In Experiment 1, washed sperm were re-suspended in PEY (15%) or SL (1%) media, while unwashed semen was only diluted in SL medium. In Experiment 2, washed and unwashed sperm were diluted in PEY and SL media, respectively, with there being different BHT concentrations (0.6, 2.0 and 5.0 mM). In both experiments, after 4 h of refrigeration, there were no differences neither in sperm viability nor plasma membrane functional integrity (HOST) between groups when there were evaluations using eosin-nigrosine staining. After thawing, however, there was a negative effect on motility of washed sperm preserved in SL media. Furthermore, results from cytometry evaluations indicated there was a larger population of thawed sperm with intact plasma (SYBR-14+/PI-) and acrosome (PE-PNA-) membranes, but inactive mitochondria (Mitotracker deep red-) when SL media were used. When there was BHT supplementation, there was only a slight enhancement of motility of spermatozoa preserved in PEY media with 5 mM BHT. In conclusion, when effectiveness and efficiencies are considered, PEY is the non-penetrating cryoprotectant that should be utilized for buck sperm cryopreservation.

Correlation of testicular artery Doppler velocimetry with kinetics and morphologic characteristics of epididymal sperm in dogs

Sperm quality can be affected by a reduction in testicular blood flow, which can be measured by Doppler ultrasonography. The aim of this study was to correlate the Doppler velocimetry of the testicular artery with kinetics of the epididymal spermatozoa in dogs. Twenty-two dogs (44 testicles) were evaluated by Doppler ultrasonography in five regions of the testicular artery before orchiectomy. Spermatozoa were recovered by the epididymal tail compression technique and analysed for kinetics on a computer-assisted semen analysis (CASA system). Morphology (modified Karras) and sperm membrane integrity were analysed by eosin-nigrosine staining. Data were analysed by Pearson's correlation test (p<.01). The mean total motility was 69.0%±17.7, progressive motility was 43.7%±14.7, average path velocity (VAP) was 127.0µm/s±20.7, curvilinear velocity (VCL) was 221.0µm/s±31.1, and sperm velocity index (SVI) was 389.9±56.1. There were positive correlations between the peak systolic velocity (PSV) in the proximal supratesticular region with the SVI (r=.529), VCL (r=.555) and VAP (r=.473), and a negative correlation with the percentage of slow spermatozoa (r=-.463). The results suggest that the testicular artery blood flow velocity can positively affect the speed of spermatozoa movement. For the first time, we have correlated sperm kinetics with the Doppler evaluation of the testicular artery in dogs.

A technical note on the assessment of human sperm vitality using eosin-nigrosin staining.

How many spermatozoa and slides need to be counted to make a reliable assessment of sperm vitality? Currently, various authorities recommend assessing human sperm vitality on counts of at least 200 cells, but on one or two slides. This was an observational study on duplicate eosin-nigrosin stained sperm vitality slides made from 58 ejaculates. Assessments were made using counts of 2 × 100 and 1 × 200 cells per slide, all performed by the same trained expert observer. Although assessments tend to show fewer and smaller outlier values when based on counts of 200 spermatozoa than 100, and on 2 × 200 than 1 × 200, counting 200 spermatozoa from one slide provides a result with sufficient accuracy for the clinical purpose of ascertaining whether the immotile spermatozoa in an ejaculate showing low sperm motility are alive or dead. While the increased accuracy of results derived from counts of 2 × 200 cells might be important in research studies where sperm vitality is the specific end-point of interest, counting at least 200 spermatozoa from one smear is sufficiently accurate for the clinical purpose of establishing whether the immotile spermatozoa seen in ejaculates with low sperm motility are alive or not. Consequently, the extra workload of performing replicate counts and the associated calculations does not increase the clinical value of the result, and hence is unnecessary in routine semen analysis. Careful laboratory technique as well as proper staff training and competence are essential. The conclusions might not be applicable to staining methods other than the recommended one-step eosin-nigrosin technique.

The Effect of Genistein on the Plasma Membrane Integrity of Frozen Ongole Grade Bull Semen Based on Skim Milk – Soy Lecithin Extender

Beef cattle represent livestock commodities that can help support national economic stability. Ongole grade bull (PO) is one of the local cattle breeds whose population and genetic quality decrease. Various efforts have been made in improving the genetic quality and in increasing the population of the local livestock population to achieve self-sufficient status of the beef supply in Indonesia. One of the efforts is artificial insemination (IB). The study aims at examining the effect of different concentrations of genistein semen extender on the post-thawing membrane integrity of Ongole grade bull semen. It is useful in developing the genistein as a protective agent against reactive oxygen species (ROS) during cryopreservation process. It was conducted at BPBPTDK, Center for Animal Breeding Development, Animal Feed, and Veterinary Diagnostics, in the Special Region of Yogyakarta from February 7th, 2019 to May 7th, 2019. The semen of two healthy Ongole grade bulls was consecutively collected five times in the period of 3 months using artificial vagina. The semen was cryopreserved using skim milk-soy lecithin semen based extenders at different concentrations of genistein, which were group A (1.0 mmol genistein), B (2.0 mmol genistein) and K (without any genistein as control). The observed parameters included individual spermatozoa motility, percentage of spermatozoa viability that was observed using Eosin nigrosine staining method and spermatozoa membrane integrity that was observed using the hypo osmotic swelling test (HOST) method. The observations were made on fresh semen, extended semen, and post-thawing semen. The results of the observation were then analyzed descriptively and statistically using regression correlation test and one-way analysis of variance (ANOVA). The results of the analysis showed that the addition of Genistein at the different concentrations of 1 mmol and 2 mmol resulted in higher motility, viability, and membrane integrity than the control and had significant effect (p<0.05) on the motility, the membrane integrity, and the acrosome integrity of the Ongole grade bull semen. The genistein concentration of 2.0 mmol gave the highest mean percentage of spermatozoa motility (57.50 ± 1.00 %) after freezing. The genistein concentration of 1.0 mmol gave the highest mean percentage of spermatozoa membrane integrity and acrosome integrity (52.45 ± 0.85 % and 49.58 ± 0.98 %) after freezing.

Semen bacterial contamination: Antibiotics susceptibility pattern and standard sperm parameters in men of Birjand, 2017

Background and Aim: Urinary tract infections can be one of the most important causes of infertility in men. Identification of semen bacterial contamination and using of appropriate antibiotic treatment can cause to improvement sperm parameters. The aim of this study was to evaluate the frequency of bacterial contamination of semen and its effect on standard sperm parameters. Materials and Methods: In this descriptive-analytical study, the semen analysis of 40 men referring to Birjand laboratories was performed by using the CASA model spermogram device. The Eosin-Nigrosin staining was used to evaluate of sperm viability. After semen samples were cultured on microbial culture media, bacterial contamination was assessed by using conventional microbiological methods. The determination of antibiotic susceptibility of bacterial isolates was performed by the disk diffusion method. Results: In the present study, 30% of semen 12 samples were detected with bacterial contamination, the most frequent of which was related to Escherichia coli (41.7%). Statistical analysis showed that there was a significant relationship between bacterial contamination of semen and history of urinary tract infection (UTI) (P=0.001). There was a significant decrease in the motility, viability and sperm count in subjects with bacterial semen contamination (P&lt;0.05). Nitrofurantoin, Gentamicin and third-generation of Cephalosporins were reported as the most effective treatment options for reducing bacterial contamination in semen. Conclusion: The bacterial contamination of semen can significantly reduce the motility, viability and sperm count. Due to the high prevalence of bacterial contamination in semen and its significant association with sperm factors, the microbial screening of infertile couples without clinical symptoms is essential. Key Words: Antibiotic Resistance; Bacterial Contamination; Infertility; Semen fluid; Sperm

Semen Bacterial contamination: Antibiotics susceptibility pattern and standard sperm parameters in men of Birjand,2017

Background and Aim: Urinary tract infections can be one of the most important causes of infertility in men. Identification of semen bacterial contamination and using of appropriate antibiotic treatment can cause to improvement sperm parameters. The aim of this study was to evaluate the frequency of bacterial contamination of semen and its effect on standard sperm parameters. Materials and Methods: In this descriptive-analytical study, the semen analysis of 40 men referring to Birjand laboratories was performed by using the CASA model spermogram device. The Eosin-Nigrosin staining was used to evaluate of sperm viability. After semen samples were cultured on microbial culture media, bacterial contamination was assessed by using conventional microbiological methods. The determination of antibiotic susceptibility of bacterial isolates was performed by the disk diffusion method. Results: In the present study, 30% of semen 12 sampleswere detected with bacterial contamination, the most frequent of which was related to Escherichia coli (41.7%). Statistical analysis showed that there was a significant relationship between bacterial contamination of semen and history of UTI(Urinary ??? infection) (P = 0.001). There was a significant decrease in the motility, viability and sperm count in subjects with bacterial semen contamination (P &lt;0.05). Nitrofurantoin, Gentamicin and third-generation of Cephalosporins were reported as the most effective treatment options for reducing bacterial contamination in semen. Conclusion: The bacterial contamination of semen can significantly reduce the motility, viability and sperm count. Due to the high prevalence of bacterial contamination in semen and its significant association with sperm factors, the microbial screening of infertile couples without clinical symptoms is essential. Key Words: Antibiotic Resistance; Bacterial Contamination; Infertility; Semen fluid; Sperm

Antifertility effects of Azadirachta indica methanol seed extract on canine spermatozoa in vitro

Objective: To investigate the effects of Azadirachta (A.) indica methanol seed extract on canine male fertility in vitro.Methods: The effect of the A. indica methanol seed extract (2.5, 5.0, 10.0 and 20.0 mg/mL, respectively) on canine spermatozoa was evaluated in vitro at 1.0, 2.5, 5.0 and 10.0 min post-treatment. The two controls were comprised of sperm sample in phosphate- buffered saline, and sperm sample in phosphate-buffered saline and 1% dimethyl sulfoxide, respectively. The experiment was repeated (n=3) in duplicates with semen collected from 3 adult Basenjis. The effect of A. indica methanol seed extract treatments on canine spermatozoal total and progressive motility (phase-contrast microscopy), sperm vitality (eosin-nigrosin vital staining method) and total abnormalities (phase contrast microscopy, eosin-nigrosin staining and Papanicolaou staining methods) were determined.Results: A. indica methanol seed extract treatment caused a concentration and time-dependent decrease in sperm percentage total motility, progressive motility and vitality. The absence of sperm revival post-treatment provided evidence of a permanent or irreversible incapacitation of sperm by A. indica methanol seed extract.Conclusions: The inhibition of sperm motility and significant spermicidal effect observed post-treatment suggest that A. indica methanol seed extract may produce a potent antifertility or contraceptive effect in dogs if applied as an intra-vaginal cream pre- coitus.

Relationship of frozen-thawed semen quality with the fertility rate after being distributed in the Brahman Cross Breeding Program.

Background and Aim:Various factors can reduce the quality of semen used for artificial insemination and have an impact on fertility decline, such as poor handling during frozen semen distribution. This study was aimed at assessing the quality of frozen-thawed semen after distribution in the field and its importance in maintaining fertility.Materials and Methods:The Brahman Cross (BX) breeding program of PT Lembu Jantan Perkasa, Indonesia, was used. This program was preferred due to its adherence to guidelines that limit the effects of extraneous factors that may affect semen quality. Frozen-thawed semen samples from eight bulls with the same production code were analyzed and compared between the production site (artificial insemination [AI] center) and the field (BX breeding program). Total and progressive motility (PM) of sperm were determined using computer-assisted semen analysis. Plasma membrane integrity (PMI) was assessed using hypoosmotic swelling test, sperm viability using Eosin-Nigrosin staining, acrosome integrity using trypan blue-Giemsa staining, morphological abnormalities using William staining, and DNA fragmentation using toluidine blue staining. The fertility rate was determined using the conception rate (%) derived from AI data based on 502 AI services and 478 cows in the BX breeding program. A t-test was used to compare the quality of frozen-thawed semen before and after distribution. The relationship between the qualities of frozen semen after distribution in the field with fertility was analyzed using Pearson correlation.Results:There was no significant difference (p>0.05) in the quality of frozen-thawed semen (sperm motility, PMI, viability, acrosome integrity, abnormalities, and DNA fragmentation) between the production site (AI center) and after distribution in the field (BX breeding program). The semen met the minimum standards for AI programs. Total motility (r=0.986), PM (r=0.961), sperm viability (r=0.971), PMI (r=0.986), and acrosome integrity (r=0.992) were all positively correlated (p<0.05) with fertility rate; while sperm abnormalities (r=−0.996) and sperm DNA fragmentation (r=0.975) were negatively correlated (p<0.05) with fertility rate.Conclusion:The study showed that to achieve the maximal and optimal fertility rate in bulls in an AI program, the overall quality of frozen-thawed semen in all aspects is critical. This can be achieved if the handling during distribution and storage, as well as the various factors that may affect the quality of semen in the field, can be controlled properly.

Increased quality of in natura and cryopreserved semen of water buffaloes supplemented with saturated and unsaturated fatty acids from the palm oil industry.

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,ω6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P<0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P<0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P<0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P<0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.

The effect of royal jelly on boar sperm viability and motility during liquid storage for 96 hours

The current study was carried out to investigate the protective effects of royal jelly supplementation on sperm motility, viability and pH value during the liquid storage of boar semen at 16 °C and 4 °C, at various periods of time (0, 24, 48, 72 and 96 h). Semen samples were collected from 11 boars, diluted with a long-term extender and supplemented with different concentration of royal jelly (0%, 0.5%, 1% and 2%) at a final concentration of 50 × 106sperm/ml. In the laboratory, the semen was assessed for sperm morphology, viability (eosin-nigrosin staining), subjective motility and objective sperm motility by sperm class analyzer. In total, 396 tests for sperm viability and motility were performed. The longer storage time and the lower incubation temperature showed lower sperm motility and viability results. The results showed that royal jelly supplementation at 1% concentrations protected the functionality of the sperm plasma membrane during the liquid storage time of 96 h at 16 °C. Sperm subjective and objective motility results in samples stored at 4 °C decreased with higher royal jelly concentrations and longer storage time, and differ significantly from the results in samples stored at 16 °C (P&lt; 0.05). Our data showed that royal jelly supplementation at lower concentrations can improve boar semen motility and viability parameters during liquid storage at 16 °C for 96 h.

The in vitro effect of kanamycin on the behaviour of bovine spermatozoa

The use of antibiotics is a common part of animal biotechnologies. Especially, the use of antibiotics in semen extenders is necessary. However, the effect of antibiotics on the spermatozoa structure and function is still not completely examined. Therefore, the aim of our study was to investigate the effect of kanamycin on bovine spermatozoa at concentrations of 80 and 160 µg/mL during the 24 h in vitro cultivation. Semen samples were collected from clinically healthy Holstein-Friesian bulls. At times of 0, 2 and 24 h the motility assessment, mitochondrial activity, acrosomal and membrane integrity evaluation were performed. The sperm motility was measured using the Computer-assisted sperm analysis (CASA). Mitochondrial activity was evaluated through the Mitochondrial Toxicity Test (MTT). The acrosomal status was determined using the fast green/rose bengal staining on slides. Similarly, the membrane integrity was analysed using the eosin-nigrosin staining. Our results revealed the dose- and time-dependent effect of kanamycin under the in vitro conditions. In conclusion, the selected concentrations of kanamycin may have adverse effects on the motility, mitochondrial activity, acrosomal and membrane integrity during semen processing. Considering the relatively low concentrations used, we do not recommend to use kanamycin as a supplement in bovine semen extenders.

Influence of Coconut Powder Water-Based Conservation Medium (APC-102c) for Maintaining Mitochondrial Activity of Cryopreserved Ram Sperm

Background: Semen extenders are required to protect and preserve semen, and the development of suitable extenders is key for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is through microscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and may not provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to add reliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a more accurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods &amp; Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10). Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS + 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through the refrigeration curve up to 4°C (0.35° C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cm above liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Both fresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x), and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with the Eosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained). Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3'-diaminobenzidine (DAB); 200 sperms were counted, and classified into four classes (I, II, III, and IV) according to the degree of coloration of the intermediate part. Fresh semen showed no significant difference (P &gt; 0.05) between treatments with respect to motility parameters; however, T2 showed significantly inferior results regarding plasma membrane integrity. After thawing, T2 was significantly higher in sperm motility parameters compared to T1. The mitochondrial activity and plasma membrane integrity parameters did not show any significant difference between the treatments.Discussion: The TRIS-based diluent showed higher motility values than ACP-102c; however, motility rates in ACP-102c diluent, although lower, are considered satisfactory for insemination, which requires semen with minimal progressive motility of 30%. Notably, the cryopreservation protocol used in this study is the standard for TRIS-based diluent, and it is known that the optimal rate of refrigeration and cryopreservation may differ according to the composition of the storage medium; therefore, we may assume that the protocol used is not yet appropriate for the ACP-102c diluent, and further studies are required. IMP is an essential attribute for fertilization, and cryopreservation can affect the plasma membrane as observed in this study. Cryopreserved semen reduced the percentage of class I mitochondrial reaction sperms in both treatments, demonstrating that cryopreservation affects the mitochondrial activity of the intermediate portion of the sperm; however, there was no difference between treatments in thawed semen. Thus, we concluded that the ACP-102c conservation medium maintains seminal quality after thawing, and it can be used in artificial insemination processes.

Intervals of Semen Collection from Boars during Early Reproductive Life: Evaluation of Seminal Fluid Parameters

Background: In swine production, good reproduction rates can be achieved through genetic selection and reproductive biotechnologies. One of these biotechnologies is artificial insemination, which contributes to disseminate genes and optimize breeding boars, thus improving the quality of insemination doses. This study focused on evaluating the intervals between semen collection from boars at the beginning of their reproductive maturity vis-à-vis the viability of insemination doses. Materials, Methods &amp; Results: Twenty 9-month-old boars of the genetic lineage AGPIC 337 (Agroceres PIC) were used in this study. The experimental design used here was completely randomized, and the randomly selected males were divided into four treatment groups, which were named according to the interval between semen sample collections: T1: 2 days; T2: 3 days; T3: 4 days and T4: 7 days. Each treatment comprised 5 animals, and at the end of the 90 days of this study, a total of 150 ejaculates were obtained in T1, 110 in T2, 90 in T3 and 60 in T4. The values of total motility, volume and sperm concentration of the ejaculates were evaluated, as was oxidative stress by means of thiobarbituric acid reactive substances (TBARS), the latter after 0, 72, 120 and 168 h of the study. Membrane integrity was evaluated at 0, 72, 120 and 168 h using the eosin-nigrosin staining procedure. Sperm heat resistance was tested after 120 h, and sperm morphology after 72, 120 and 168 h. Sperm concentrations differed, with T3 showing 27.04% and 29.65% higher concentrations (P &lt; 0.05) than groups T2 and T1, respectively. Total motility in group T4 was 0.56%, 1.98% and 3.28% higher (P &lt; 0.05) than in T3, T2 and T1, respectively, indicating that the 7-day interval produced the best result. The heat resistance test showed the expected results, i.e., T2 and T4 did not differ in terms of total motility, but that of T4 was 4.96% higher (P &lt; 0.05) than T3 and 7.71% higher than T1. As for plasma membrane integrity, T4 had 6.45%, 8.09% and 12.72% more cells with intact membranes (P &lt; 0.05) than T3, T2 and T1, respectively. With regard to TBARS, group T1 showed higher concentrations (P &lt; 0.05) than T2 (6.18%), T3 (11.13%) and T4 (9.12%), i.e., the sperm cells in the animals of T1 showed greater changes. As for sperm morphology, the number of intact cells in T4 was 6.08%, 7.53% and 12.44% higher (P &lt; 0.05) than in T3, T2 and T1, respectively. Discussion: With respect to total motility, other authors have reported similar results, i.e., the motility of sperm was highest when collected at 7-day intervals and lower when collected at shorter intervals. Despite the statistical differences between treatments, these results were found to be similar to those recommended in the literature for use in the preparation of insemination doses. Thus, we suggest that all the intervals between collections can be used in the routine of semen production centers, since the minimum requirement for an ejaculate to be accepted in an artificial insemination program is to have a total motility of no less than 70 percent. At the beginning of their reproductive maturity, boars may be subjected to intervals between collections of at least 2 days, without impairing the total motility of insemination doses. However, the best results are obtained when semen is collected at 7-day intervals, which ensures higher total motility, morphologically healthy cells with intact membranes, and less oxidative damage caused by lipid peroxidation.

26 Baobab oil supplemented extender preserves post-thaw bull sperm quality parameters

The processes of semen cryopreservation and thawing affect sperm membrane integrity and motility and increases morphological defects as well as DNA damage. The most influential cause of this is oxidative stress. When endogenous antioxidant capacity of seminal plasma is reduced during the freeze-thawing process, plant extracts exhibiting strong antioxidant activity can be used as supplements for compensation. Baobab oil has gained interest because it is rich in powerful antioxidants, which could protect sperm cells from oxidative damage during cryopreservation. Our study aimed to assess the effects of baobab oil on post-thaw sperm quality parameters in an egg-yolk-based extender. Thirty semen ejaculates were collected from 15 Nguni bulls using an electro ejaculator. Semen samples were randomly allocated to control (no baobab oil), 20μL (1%), 50μL (2.5%), and 100μL (5%) baobab oil per millilitre extender. Following dilution, semen samples were loaded into 0.25-mL semen straws, equilibrated for 4h at 5°C, and transferred into a controlled rate programmable freezer. The frozen semen straws were stored in a liquid nitrogen tank (−196°C) until thawing. Semen straws were thawed (37°C/60 minutes) after 1 week of cryopreservation and analysed for (1) sperm motility using a computer-aided sperm analyser, (2) morphological defects and viability using eosin-nigrosin stain, (3) membrane integrity by hypo-osmotic swelling test, and (4) DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. Data was analysed using analysis of variance. Treatment means were compared in relation to the control group by Dunnett's test. We found that supplementing semen extender with baobab oil at 1% significantly (P&amp;lt;0.05) preserved sperm DNA integrity (88.3±3.7) and membrane integrity (74.0±4.2) when compared with the control group (71.7±3.7 and 55.8±4.4, respectively). Baobab oil supplementation either at 1% (5.9±0.5), 2.5% (7.2±0.5), or 5% (6.0±0.5) significantly reduced sperm morphological defects compared with control (9.5±0.5). Total motility (1% (72.7%), 2.5% (72.7%), 5% (71.9%), control (59.3%)) and viability (1% (79.1%), 2.5% (79.8%), 5% (77.8%), control (67.6%)) were also improved by supplementation; however, the difference was not significant. In conclusion, it was demonstrated that supplementing bull semen extender with 1% baobab oil protects sperm from morphological defects, maintains membrane integrity, as well as preserves sperm DNA. All the baobab oil supplementation levels preserved post-thaw bull-sperm quality parameters.