Sperm Diluents Research Articles

Comparative study of Tris Turmeric, Tris Turmeric Dimethyl Sulfoxide and Tris Turmeric Ethylene glycol extenders on the cryosurvivability, sperm resistance, in- vivo fertility and antioxidant status in buffalo bull semen

The freeze-thaw process leads to structural and functional damage due to excessive accumulation of reactive oxygen species (ROS). The addition of exogenous antioxidants to sperm diluents is of great importance to overcome oxidative damage during freezing. The purpose of this study was to explore the effects of three diluents Tris Turmeric, Tris Turmeric Dimethyl Sulfoxide and Tris Turmeric Ethylene glycol on the cold survival ability of buffalo sperm. Semen was collected from five local adult male buffalo breeds. A base diluent of Tris-citric acid-fructose (TCF) was prepared, adding 20% whole egg yolk (TCFY). The Tris extender without turmeric, without DMSO, and without EG was kept as a control. Other extenders are Tris containing turmeric TT (100 ml/5 ml Tris), Tris containing turmeric dimethyl sulfoxide TTD (100 ml/5 ml Tris + 1.5% DMSO) and Tris containing turmeric and ethylene glycol TTE EG (100 ml/5 ml Tris + 1.5% EG). Semen samples were added and a pure sperm concentration of 60 × 106/ml was achieved. Frozen buffalo sperm after thawing showed significant improvements in all research parameters of the three breeding samples compared to the control. Tris Turmeric Ethylene was the type that best improved sperm survival under frozen conditions, followed by Tris Turmeric and Tris Turmeric Dimethyl Sulfoxide compared to the control. A significant decrease in sperm motility after thawing was evident as usage time increased in all expanders. There was a significant increase in total antioxidant content (TAC) and insignificant change in malondialdehyde (MDA) of the diluent used compared to the control. Conception rate (CR) was higher in Tris Turmeric Ethylene glycol (65.2%), followed by Tris Turmeric (60.3%) and Tris Turmeric Dimethyl Sulfoxide (55.9%) compared to the control (36, 7%). It can be concluded that Tris Turmeric Ethylene Glycol is considered the best agent for improving cold survival and sperm fertility, followed by Tris Turmeric and Tris Turmeric Dimethyl Sulfoxide.

Conserving goat sperm post-thawed gene expression and cellular characteristics using the antioxidant coenzyme Q10 supplementation.

The use of frozen goat semen for artificial insemination frequently results in a decline in sperm quality following thawing, which can be attributed to cold shock from cryopreservation, reduced motility, and possible DNA damage. Freezing may compromise mRNA stability due to the presence of free radicals. Despite strong post-thaw motility and no visible DNA fragmentation, sperm can still exhibit altered gene expression patterns. To reduce the damaging impact of free radicals during cryopreservation, antioxidants are typically added to the freezing medium. This study assessed the impact of adding coenzyme Q10 (CoQ10) to frozen sperm diluent on the ATP5F1A and CPT2 gene expression, sperm motility, and viability post-thawing. CoQ10 was added to sperm at six different concentrations: 0 mg/dL (P0), 6.25 mg/dL (P1), 12.5 mg/dL (P2), 25 mg/dL (P3), 50 mg/dL (P4), and 100 mg/dL (P5). The Statistical Package for the Social Sciences (SPSS) software version 22 was used to conduct comparative tests using one-way analysis of variance followed by Duncan's test for motility and viability and Kruskal-Wallis test followed by pairwise comparison test for membrane integrity and gene expression. The addition of CoQ10 to semen diluent has a notable impact on the post-thawed quality of sperm. The most significant outcomes were observed with a 25 mg/dL dosage (P3) for cell viability, membrane integrity, and ATP5F1A gene expression, and with a 50 mg/dL dosage (P4) for sperm motility, membrane integrity, and CPT2 gene expression. Incorporating CoQ10 into frozen semen diluent improves gene expression and prevents deterioration of the cell quality of thawed goat spermatozoa. While the study demonstrates the benefits of CoQ10, the precise molecular mechanisms through which CoQ10 enhances gene expression and cell quality were not fully elucidated. Further investigation is needed to understand these mechanisms in detail. Comparative studies with other antioxidants and cryoprotectants can help establish the relative efficacy of CoQ10 and potentially develop more effective combinations.

Positive In Vitro Effect of ROCK Pathway Inhibitor Y-27632 on Qualitative Characteristics of Goat Sperm Stored at Low Temperatures.

Y-27632, as a cytoskeleton protector, is commonly used for low-temperature preservation of cells. Goat sperm are prone to damage to the cytoskeleton under low-temperature conditions, leading to a loss of sperm vitality. However, the Y-27632 small molecule has not yet been used in research on low-temperature preservation of goat semen. This study aims to address the issue of low temperature-induced loss of sperm motility in goats by using Y-27632, and explore the regulation of Y-27632 on goat sperm metabolism. At a low temperature of 4 °C, different concentrations of Y-27632 were added to the sperm diluent. The regulation of Y-27632 on the quality of low temperature-preserved goat semen was evaluated by detecting goat sperm motility, antioxidant capacity, mitochondrial activity, cholesterol levels, and metabolomics analysis. The results indicated that 20 µM Y-27632 significantly increased plasma membrane integrity (p < 0.05), and acrosome integrity (p < 0.05) and sperm motility (p < 0.05), increased levels of superoxide dismutase (SOD) and catalase (CAT) (p < 0.01), increased total antioxidant capacity (T-AOC) (p < 0.05), decreased levels of malondialdehyde (MDA) and reactive oxygen species (ROS) (p < 0.01), and significantly increased mitochondrial membrane potential (MMP). The levels of ATP, Ca2+, and TC in sperm increased (p < 0.01). Twenty metabolites with significant differences were identified, with six metabolic pathways having a significant impact, among which the D-glutamic acid and D-glutamine metabolic pathways had the most significant impact. The artificial insemination effect of goat semen treated with 20 μM Y-27632 was not significantly different from that of fresh semen. This study indicates that Y-27632 improves the quality of low-temperature preservation of sperm by protecting the sperm plasma membrane, enhancing sperm antioxidant capacity, regulating D-glutamine and D-glutamate metabolism, and promoting the application of low-temperature preservation of semen in artificial insemination technology.

Review Jurnal: Efektivitas Penggunaan Kuning Telur Berbagai Jenis Unggas Sebagai Pengencer Semen pada Ternak

A method is needed that can be used to maintain sperm quality in order to produce superior seeds. The use of poultry egg yolk as a diluent is of course widely used because poultry eggs can maintain the good quality of cattle sperm. This research aims to determine the level of effectiveness of egg yolks of various types of poultry as a sperm diluent on the motility and viability of cattle sperm. The method used in this research is the literature review method and the descriptive method. The results showed that the use of free-range chicken egg yolk and quail egg yolk as sperm diluent showed the highest motility values. The highest percentage of viability was in purebred chickens, while the lowest abnormality was in dilution with goose yolk eggs.

The cryoprotective effect of Guar gum co-supplemented with ethylene glycol and glycerol in Simmental bull semen

Sperm cryopreservation often reduces sperm quality by forming of intra- and extracellular ice crystals. Various compounds widely used to counteract this effect. The guar gum was considered as an extracellular cryoprotective substance. The present study evaluated the impact of the co-supplementation of guar gum with ethylene glycol or glycerol in the cryopreservation of bull sperm. Four ejaculates from 4 bulls were pooled and divided into ten groups consisting of 4 controls (glycerol 6%, ethylene glycol 6%, glycerol 3.5%, and ethylene glycol 3.5%, and six treatment groups including guar gum in 0.001% and 0.002% alone and or co-supplemented either with 3.5% glycerol or 3.5% ethylene glycol and frozen in liquid nitrogen. The sperm motility, viability, plasma membrane and DNA integrity, apoptotic-like changes, antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities evaluated. The groups contained 3.5% glycerol + 0.001% guar gum, 3.5% ethylene glycol + 0.001% guar gum, and 0.001% guar gum alone showed higher values for live sperm, antioxidant enzymes, membrane integrity, mitochondrial membrane potential (MMP), fertilization, cleavage, and blastocyst rates; and lower values for apoptotic-like changes, H2O2 level, and DNA damage than the control groups. In conclusion, adding guar gum to the bull sperm diluent either alone or combined with glycerol or ethylene glycol ameliorated sperm viability and kinematic parameters and antioxidant capacity while reducing DNA damage and apoptotic-like changes. Guar gum also has improved embryo development. Due to its cost-effectiveness and physicochemical properties, guar gum is a promising supplement for bull sperm cryopreservation.

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.

The importance of sperm diluents of different types of farm animals in the process of the cryopreservation

Purpose:to systematize and analyze scientific information about diluents of sperm of farm animals in the process of its cryopreservation, taking into account specific featuresAssisted reproductive technologies are extremely in demand in animal husbandry at the moment, especially with regard to artificial insemination. However, this technology is of priority importance if the sperm is stored for a long period when the sperm is cryopreserved. At the same time, numerous studies have established that the cryopreservation process causes adverse changes in germ cells, which leads to their damage or death, and partially irreversible damage to the morphological structures of cells occurs. To obtain high results from the use of frozen-thawed sperm, it is necessary to prevent damage to spermatozoa. In this regard, the use of sperm diluents is urgent. Each type of animal sperm has its own distinctive parameters volume, concentration, chemical composition, osmotic pressure, pH, etc. Therefore, it is difficult to develop a universal sperm diluent suitable for each type of animal.

36 Evaluating the toxicity of several sperm diluents over time and at varying osmolalities in an internally fertilising salamander species

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Modern technologies for storing semen of domestic animals without the addi-tion of antibiotics

Currently, there is a trend of breeding domestic animals through artificial insemination. As a result, very large amounts of sperm diluents containing antibiotics are used in animal husbandry. Antimicrobials are added to the semen diluent to control the growth of bacteria that contaminate the semen during selection. The proportion of antibiotic-resistant bacteria is steadily rising, threatening the entire health care system. That is why all fields of antibiotics application face the task of finding alternatives to this approach. The purpose of our study was to systematize modern technologies and methods of storing domestic animals’ semen which could reduce or eliminate the use of antibiotics, and would be an important step in the fight against multidrug-resistant bacteria. Due to the negative impact of antibiotics on sperm quality and their fertilizing ability, new alternative methods for sperm storage are constantly being improved and developed. The most common are low-temperature storage, physical methods to reduce bacterial stress, the use of antimicrobial peptides, nanoparticles and the use of various substances of animal, plant or other origin. The possibility of boar sperm low-temperature storage may open up completely new approaches in the future by optimizing the cooling rate. Colloidal centrifugation as one of the physical methods is a practical means of reducing the bacterial load in sperm samples and it can be effectively used applying equipment that is available at many breeding plants. Antimicrobial peptides or nanoparticles of iron oxide may be a useful alternative to the addition of antibiotics during sperm storage. Antimicrobial peptides have been shown to control the growth of aerobic and anaerobic bacteria in relatively low concentrations without adversely affecting sperm quality and fertility. However, it is substantiated that nanoparticles with the size of 40 – 60 nm have significant antimicrobial ability against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. At the same time, further studies are needed on the use of various substances of animal or plant origin (royal jelly, aloe vera, algae extracts), as well as determining adequate concentrations of these new compounds that should be effective in fighting bacteria and not affect quality characteristics of sperm.

The effect of vitamin e antioxidant addition in goat milk diluent on the quality of thin-tailed sheep semen

Artificial insemination supports the prevention of infectious dispersion in organisms which share the same environment. Meanwhile, spermatozoa usually experience a decrease in quality during processing due to the peroxidation process. This study aims to identify the effect of vitamin E antioxidant addition in goat milk diluent on the quality of thin-tailed sheep semen after storing at 5°C for five days. A total of 4 male thin-tailed sheep were used and the treatments consisted of controls, namely goat milk sperm diluent without vitamin E antioxidant (P0), and others with the addition of vitamin E at a dose of 2 gL-1 diluent (P1); 3 gL-1diluent (P2); and 4 gL-1 (P3). Moreover, the variables observed include individual motility, viability, and intact plasma membrane of spermatozoa after storing at 5°C for five days. The data were analyzed statistically using analysis of variance (ANOVA) with a completely randomized design in one-way. The results showed that the addition of vitamin E antioxidant to goat milk diluent during sperm dilution had a significant effect (P < 0.05) on the motility, viability, and intact plasma membrane. Spermatozoa motility at P0, P1, P2, and P3 were 44.0%, 48.2%, 50.4%, and 53.2%, while the viability was 48.2%, 51.4%, 54.2%, and 56.0%, respectively. Additionally, the intact plasma membranes at P0, P1, P2, and P3 were 41.0%, 46.6%, 48.2%, and 51.4%, respectively. The addition of antioxidant vitamins in goat milk diluent with a concentration of 4 gL-1 produced the best results on sperm quality after cooling. Based on the results, it was concluded that the addition of vitamin E antioxidant to the sperm diluent can improve the quality of thin-tailed sheep chilled semen.

Boar Semen Contamination: Identification of Gram-Negative Bacteria and Antimicrobial Resistance Profile

Simple SummaryBoar semen can contain many bacterial species, some of which can have a negative impact upon the quality of the semen, as well as on the sows’ reproductive capacity. Semen contamination may occur at time of collection or during semen processing. The aim of this study was to identify gram-negative bacteria that appear in boar semen and to establish models of antimicrobial resistance of isolated gram-negative bacteria. Semen doses examined contained bacterial species with a known negative effect on sows’ reproductive tracts (Pseudomonas, Enterobacter, Klebsiella, E. coli), and more than half of these isolates were resistance to gentamycin (56.52%) and penicillin (58.69%) antimicrobials commonly used in boar semen extenders. This work proved the presence of pathogenic multiple resistant bacteria in semen, and therefore, we highly recommend periodic microbiological screening of bacteriospermia in boars to avoid the use of low-quality semen in the pig industry.Bacterial contamination of boar semen occurs with some frequency in artificial insemination centers and may have a negative effect on the quality of the semen as well as on the sows’ reproductive capacity. Normally, the source of bacterial contamination in pig seminal doses is the own boar. However, distilled water or laboratory equipment used to elaborate the seminal doses can be an important source of bacterial contamination. This study focused on the identification of gram-negative bacteria in boar semen, and impact on the quality of ejaculates obtained from boar, as well as on the establishment of antimicrobial resistance patterns of isolated gram-negative bacteria. Semen samples were collected from 96 boars, ranging in age from 12–36 month, from three artificial insemination centers from the North-West of Romania. Bacterial species were identified by two methods: matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and API 20 E (BioMerieux, France). The main bacteria isolated from the doses diluted semen were gram-negative bacteria (47.91%), with a majority of the contaminant bacteria belonging to the family Enterobacteriaceae: Seratia marcescens 19.56%, Proteus mirabilis 15.21% and Escherichia coli 10.86% and to the family Pseudomonaceae: Ralstonia picketii 17.39%, Burkholderia cepacia 10.86%, Pseudomonas aeruginosa 8.69%, and Pseudomonas fluorescens 4.34%, respectively. More than half of these isolates (56.52%) were resistant to gentamycin and 58.69% were resistant to penicillin. These antibiotics are very frequently added in sperm diluent in the centers for the processing of sperm from boars in Romania. Regular monitoring for bacterial contamination is an important aspect of a control program.

Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging.

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

مطالعه تاثیر آنتی اکسیدانی عصاره هیدورالکلی درمنه (Artemisia incana) بر کیفیت اسپرم منجمد-یخ گشایی شده قوچ مغانی

Maintaining fertility in frozen sperm is very important. To investigate the effect of Artemisia (Artemisia incana) extract on sperm cryopservability of Moghani ram spermatozoa, different levels of the plant extract (0, 2, 4, 6, 8, 10 and 12 ml per deciliter) were added after extraction to diluted semen with an extender containing citrate buffer. After chilling, the semen was packaged in 0.5 ml straws and stored inside the liquid nitrogen until the evaluation of qualitative properties. This experiment was designed with 4 replications ­(with 10 straws in each replicate) per experimental group. Evaluation of sperm viability using eosin-nigrosine staining showed that adding 4 ml of Artemisia extract per deciliter to sheep sperm diluent had a positive effect on sperm viability. Similarly, the HOST test (hypoosmotic swelling test) to examine the integrity of the plasma membrane showed that the treatment had the highest membrane integrity (P<0.05). The highest and lowest Total Motility (TM) was observed in experimental groups 4 and 10 ml of extract in diluted semen, respectively (P<0.05). Examination of peroxidative damage by measuring malondialdehyde showed the lowest amount of malondialdehyde production was seen in the treatment with 12 mg of extract per deciliter diluent that was significantly different from other experimental groups (P<0.05). According to the results of the present experiment, it seems that the use of 4 ml Artemisia extract per deciliter of the diluent has positive effects on the quality of sperm after freezing.

Study of different levels of Vitamin A supplementation in extender on sperm quality in cooling storing and cryopreservation condition in Ghezel ram

Aim: This study aimed to investigate the protective effects of different levels of vitamin A on ram sperm diluent during storage until equilibrium and before cryopreservation at 4 °C and freeze-thawing. Material and Methods: In this study, 4 Ghezel rams were used for semen collection by the artificial vagina. After the initial evaluation, the samples with normal properties were pooled and after the dilution process, 0, 5, 15, and 20 µmol vitamin A were added to the samples and frozen after 2 hours of cooling. Parameters of motility, viability, plasma membrane integrity, and sperm morphology were evaluated in two steps (before and after cryopreservation) as well as malondialdehyde levels after freeze-thawing. Results: The results showed that the addition of different levels of vitamin A to the extender during cooling at 4 °C had no significant effect on the evaluated parameters (P <0.05). The results after freezing showed that a level of 15 µmol of vitamin A increased the total motility, progressive motility (PM), VAP, and VSL (P <0.05). Plasma membrane integrity and viability increased significantly at the level of 15 µmol and also adding 15 µmol levels to the freezing medium decreased the amount of sperm with abnormal morphology and the amount of malondialdehyde compared to the control group (P <0.05). Conclusion: According to the results of adding different levels of vitamin A, the level of 15 µmol has the best performance in improving the evaluated parameters and reduces oxidative stress during cryopreservation and thawing.

Economic Aspects and Evaluation of the Viability Period of Immunosexed and Diluted Ram Sperm in Powdered Coconut Water Conservation Extender (ACP-102c)

Background: Sperm sexing is increasing in use because pre-determining the sex of the calf allows greater profitability and promotes significant gains in the productive systems that utilize the technique. Deployment of a low-cost and practical preservation methodology may further favor the cost-benefit ratio. Flow cytometry, the most commonly used sexing technique, has high costs and is very restricted. As an alternative, immunosexing has been studied, which uses sex-specific monoclonal antibodies. Thus, the objective of this study was to evaluate the immunosexing technique in conjunction with cryopreservation in ACP-102c and examine its economic aspects with regard to ram semen.Materials, Methods &amp; Results: Ejaculates from two ram individuals were collected with the aid of an artificial vagina, evaluated, and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro-RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP (ACP-102c + 20% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated to 4°C, stabilized for 30 min, frozen in liquid nitrogen vapors (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were thawed and evaluated for sperm kinetics both by using computerized semen analysis with SCA® software (Sperm Class Analyzer version 5.0) and subjectively comparing specimens from the two animals using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and normal spermatozoa were determined. A bibliographic survey and a market study of similar products and technologies were carried out to provide an economic viability metanalysis of the bioproduct (ACP-102c) and bioprocess (immunosex). The data were analyzed using R-project©, and comparisons made between animals and between thawing periods using T test. There were no statistically significant differences between animals and between periods (P &gt; 0.05), except for the normal sperm parameter, in which animal A1 had the lowest percentage (P &lt; 0.05). As for the cost-benefit ratio, flow cytometry as a technique is more laborious and expensive, while immunosexing associated with cryopreservation in ACP-102c diluent has proven more practical, with regards to both sperm sexing techniques and diluents for sperm conservation.Discussion: In general, the quality of cryopreserved sexed semen was lower than that of non-sexed semen; however, in this study, both in the comparison between animals and between evaluation periods, similar values of motility, viability, and sperm morphology were obtained for sexed and several non-sexed cryopreserved semen samples, demonstrating that immunosexing did not severely affect the sperm structure, and that the ACP-102c conservation medium was efficient at maintaining the plasma membrane of these sperm. In the evaluation of economic aspects, it was observed that immunosexing, associated with cryopreservation in ACP-102c diluent, proved to be the most practical technique, requiring only conventional equipment, and allowing a greater field of application, since the immunosexing semen can be used for primiparous and multiparous females. Thus, it was concluded that immunosexing associated with cryopreservation in an ACP-102c diluent was more cost-effective, more practical, and had significantly improved sperm quality results after sexing.

Penambahan madu dalam pengenceran sperma terhadap motilitas spermatozoa, fertilisasi dan daya tetas telur ikan Patin Siam, Pangasius hipophthalmus

This research aimed to determine the optimal doses of honey addition on the sperm dilution to optimize spermatozoa motility, fertilization and hatchability of Patin Siam eggs (Pangasius hypophthalmus). The experimental design used was Complete Random Design (RAL). Diluent solution consisted of a physiological NaCl 0.9% and honey. Treatment doses of honey in aqueous diluent sperm tested as treatments were A (0 mL), B (0.2 mL), C (0.4 mL) and D (0.6 mL), each with three replications. Observations made included spermatozoa motility, fertilization and eggs hatchability. The research results showed that the addition of honey had a significant influence on motility, fertilization, and eggs hatchability. This study concluded that the addition of honey in sperm diluent solution with a dose of 0.6 mL of honey in 99.4 mL of NaCl could increase the spermatozoa motility and fertilization of Patin siam. Keywords: honey, motility, fertilization, hatchability, Pangasius hipophthalmus

Fertility disturbances of dimethylacetamide and glycerol in rooster sperm diluents: Discrimination among effects produced pre and post freezing-thawing process

With avian sperm cryopreservation protocols, the most widely used cryoprotectants (CPAs) are the glycerol (GLY; in gradual freezing: in-straw freezing method), and the dimethylacetamide (DMA; in pellets by plunging into liquid nitrogen: in-pellet rapid freezing method). Use of both methods results in a small portion of thawed live sperm with lesser fertilizing ability compared with the semen samples immediately after collection. This study was conducted to assess the pre-freezing damage occurring to the sperm due to the interaction with the cryoprotectants (CPAs) GLY (8%) and DMA (5%), as well as the post-freezing damage resulting from both freezing methods Data for each treatment, in fresh and frozen-thawed samples, were compared for sperm motility, fertilizing capacity and sperm-egg penetration holes/germinal disc (SP holes/GD). Hens (n=50) were artificially inseminated (10 hens/treatment) six times with 3day intervals between inseminations. The treatment of fresh sperm with DMA led to a reduction (P<0.05) in the count of SP holes/GD (21.4) and the fertility rate (66.7%). The addition and elimination of GLY in fresh samples resulted in a lesser (P<0.05) number of SP holes/GD (11.8) and the fertility rate (i.e., 50.0%). The number of SP-holes/GD was least in frozen-thawed samples using both DMA and GLY (14.2 and 9.2, respectively). The fertility rate when using semen frozen with DMA in- pellets was greater (P<0.05) than with use of semen that had been frozen using GLY in straws (46.4% compared with 31.3%). The reduction in fertility compared with the control when semen was cryopreserved using GLY was 64.1%; the GLY addition and elimination was responsible for two thirds of this reduction. The reduction in fertility when using semen cryopreserved with DMA was 46.7%; half of the reduction was attributed to the treatment with DMA. In conclusion, the mechanical damage attributed to the process for reducing GLY concentrations was more harmful to sperm fertilizing capacity than the toxicity of DMA and freeze/thaw process. For both freezing methods, the amount of sperm cryo-damage was similar, when the damage attributed to the CPA addition and elimination process was excluded.

"Penambahan Astaxanthin pada Pengencer Kuning Telur Berbagai Jenis Unggas Dapat Memproteksi Semen Babi Selama Penyimpanan (THE ADDITION OF ASTAXANTHIN ON SPERM DILUENTS PHOSPHATE EGGYOLK OF VARIOUS POULTRY CAN PROTECT QUALITY OF PIG SPERM DURING STORAGE) "

A study was conducted to formulate semen diluent of pigswith a better quality, cheap and easy toprepare using egg yolk of various poultries such as chickens, ducks and quails in combinationwithastaxanthin, a potent antioxidant. The research design used was a completely randomized factorialdesign with three different types of sperm diluents and four levels of astaxanthin concentration. Spermdiluents used were phosphate duck egg yolk, phosphate quail egg yolk and yolks phosphate supplementedrespectivelywith 0,002%, 0,004% and 0,008%astaxanthine. The treated sperm were strored at 5oC for 48hours. The sperm qualities were examined for progressive motility, spermatozoa abnormalities, viabilityand plasma membrane integrity. The result showed that sperm diluents of using duck egg yolk phosphatein combination with 0.002% astaxanthinresulted in the highest progressive motility, viability and plasmamembrane intact while abnormalities spermatozoa is lowest. It can be concluded that phosphate duck eggyolk sperm diluents with the addition of 0,002% astaxanthinappeared to be able to maintain the qualityof pig sperm stored at 5oC for 48 hours.

Sperm–egg penetration assay assessment of the contraceptive effects of glycerol and egg yolk in rooster sperm diluents

Glycerol (GLY) and egg yolk (EY) are good cryoprotectants of avian and mammalian sperm, but in birds, they strongly inhibit the eventual fertilization of ova. Using the sperm penetration (SP-holes) assay and fertility trials, the present study investigates (1) the possible mechanism by which this contraceptive effect occurs in chickens and (2) the maximum concentrations of GLY and EY tolerated by fresh rooster sperm. Seventy Black-Barred Andaluza hens (five per treatment) were inseminated four times (twice per week) with 0.1 mL of fresh semen from roosters of the same breed diluted 1:1 (v:v) with Lake and Ravie medium containing different concentrations of GLY or EY. No adverse effects on acrosome integrity, sperm motility, or viability were seen with any concentration of GLY or EY. The number of SP-holes on perivitelline layer samples taken from above the germinal disc became progressively lower at GLY concentrations of 1.5% or greater (P > 0.05). No holes caused by sperms were seen in unfertilized eggs. The corresponding fertility results showed similar reductions when the GLY concentration was 1.5% or greater. No changes in the number of SP-holes were seen with increasing EY concentrations (0%–7.5%), nor were any differences in fertility observed, except for a reduction when 15% EY was used. The results therefore reveal that GLY affects the transit of sperms through the oviduct in their attempt to reach the infundibulum area, limiting their access to the ovum perivitelline layer. Egg yolk had no such effect, nor did it influence acrosome reaction capacity; its mechanism of contraceptive action therefore remains unknown. The maximum GLY and EY concentrations tolerated by the rooster sperm were 0.75% and 7.5%, respectively.

Reproductive biotechnology in animal husbandry - current status and future prospects

To date there is still no optimal biotechnology which ensures maximum preservation of the functional parameters of the spermatozoa from buffaloes, boars and dogs. The aim of this research is to study the biological potential of seminal plasma proteins that are specific only to ejaculates with high cryotolerance and good quality parameters of the spermatozoa. The motility and velocity parameters of the spermatozoa were assessed by computer-assisted sperm analysis. Seminal plasma proteins were separated by size-exclusion liquid chromatography and characterized by polyacrylamide gel electrophoresis and mass spectrometry. Based on the results obtained, sperm diluents and methods for biological evaluation of the fertilization potential of the spermatozoa from buffalo bulls, boars and dogs were created and proposed for practical application.