Semen Extender Research Articles

A novel method for isolation of functional stallion spermatozoa for artificial insemination

In vitro semen purification techniques have been developed that seek to mimic the in vivo selection process, in order to generate the highest possible chance of oocyte fertilization following artificial insemination (Morrell and Rodriguez-Martinez. Vet Med Int. 2011:894767). Since this practice began, there have been many methods developed in an attempt to isolate functional spermatozoa for AI, yet only one method is routinely used – density gradient centrifugation using commercial preparations such as EquiPure. Here, we have developed a novel method of isolating functional spermatozoa and compared sperm quality to spermatozoa isolated with EquiPure. The AI-Port system (Memphasys, Sydney, Australia), comprises a disposable cartridge consisting of a sample chamber – where extended semen is deposited, and a harvest chamber – where isolated spermatozoa is extracted. These chambers are separated by a 5 µm nylon woven membrane, through which highly motile spermatozoa are able to swim from the sample chamber into the harvest chamber over 20 min, leaving behind seminal plasma and other cell types such as leukocytes. Spermatozoa were collected from miniature pony stallions, and immediately extended with EquiPlus semen extender. The ejaculate was split, and spermatozoa were isolated into BWW media with EquiPure (as per manufacturer's instructions; Nidacon, International), or the AI-Port system. Comparisons were conducted between spermatozoa isolated with the AI-Port and EquiPure, which revealed that the yield of AI-Port samples was significantly lower (25.5±5.9%) incomparison to EquiPure samples (44.9±10.2%; Kruskal-Wallis), potentially solvable with prototype improvement. Viability (Eosin-nigrosin) of AI-Port samples was significantly higher than EquiPure samples (94.4±1.1 vs 81.5±2.1%; P≤0.01; ANOVA; respectively). In regard to motility, total motility for AI-Port samples was significantly higher than EquiPure samples (93±1.3 vs 89.5±2.9%; P≤0.05; ANOVA; respectively). Whilst there was no significant difference for progressive motility between AI-Port and EquiPure samples (45.7±3.2 vs 45.6±3.4%; ANOVA; respectively). Both samples were higher than the progressive motility of the initial non-purified ejaculate (30±4.1%; P≤0.05). Mitochondrial superoxide was measured with flow cytometry and MitoSOX red. Likewise, there was no significant difference in superoxide levels between AI-Port and EquiPure samples (10.2±3.1 vs 5.6±0.8%; ANOVA; respectively). However, both samples had a reduced level of superoxide generation compared to the initial non-purified ejaculate (32.5±20.2%; P≤0.05). The DNA integrity was measured with the Halo test, which confirmed AI-Port samples had less DNA damage than EquiPure™ samples (2.0±0.5 vs 10.2±2.1%; ANOVA; P≤0.01; respectively). In summary, the AI-Port effectively isolates high-quality spermatozoa at a comparable or superior level to EquiPure. Its cost-effectiveness and user-friendliness make it a viable option for improving artificial insemination success rates in breeding programs.

Nicotinic acid addition to equine frozen semen extender influences the number of sperm bound to bovine oviduct explants

Usually, equine non-capacitated sperm bind to the isthmus region of the oviduct before ovulation occurs, a process believed essential to prepare them to fertilize the oocyte. However, during semen freezing most of the seminal plasma, a rich source of antioxidants is discarded, resulting in a higher sperm vulnerability to oxidative damage. Thus, the aim of this study was to investigate the influence of adding nicotinic acid, an antioxidant, to equine frozen semen extender to improve the binding capacity of frozen equine sperm to bovine oviduct cell explants, as an indirect way to investigate the sperm fertilizing capability. To obtain bovine oviducts, reproductive systems of abattoir slaughtered cows were transported in saline solution at 35°C to the laboratory. After sectioning the uterus-tubal junction ipsilateral to the ovaries without a corpus luteum, the isthmus region was pressed towards the uterus and its contents expelled in a Petri dish with culture medium. The oviduct contents with medium were decanted into a tube and after 5 minutes, disaggregated using a needle and syringe three times. Then, the contents were deposited in Petri dishes and cultured for 24 hours in an incubator with 5% CO2 at 38°C. Semen from ten stallions was frozen in INRA96 (control, no nicotinic acid) and with 10mM and 20mM nicotinic acid. Thawed spermatozoa(30 × 106 sperm/mL) were co-incubated with 20 oviduct explants (aggregated oviduct cells) for 30min in an incubator with 5% CO2 at 38°C. Subsequently, the explants were washed with medium to remove the loosened sperm, placed on a glass slide, and covered with a coverslip. The number of sperm bound per nm of oviduct was observed with phase contrast microscopy (40x) and counted with ImageJ software. For statistical analysis, mean and standard error were calculated. When the data did not show normal distribution, logarithmic transformation was performed and the log mean values were evaluated with variance analysis and compared with Duncan's test. The probability of P<0.05 was considered significant. The addition of 10mM nicotinic acid to equine frozen semen extender increased the number of sperm bound to the explants (79.9 ± 9.5 sperm/nm explant) compared to the control (49.1 ± 6.8 sperm/nm explant, P<0.05) and 20mM nicotinic acid treatment (65.6 ± 8.3 sperm/nm explant, P<0.05). In conclusion, the addition of 10mM nicotinic acid to equine freezing extenders improved the number of sperm bound to bovine oviduct explants. Hence, nicotinic acid may be a useful addition to freezing extenders.

Post-thaw addition of pentoxifylline to equine semen previously cryopreserved with different concentrations of cholesterol-loaded cyclodextrin

Cholesterol-loaded cyclodextrin (CLC) grants resistance to sperm during cryopreservation but may compromise sperm capacitation and fertility (Zahn et al. Theriogenology, 2002;58(2):237:240). Stimulants of sperm kinetics like pentoxifyllin (PTX) may increase sperm energy production and stimulate metabolic pathways responsible for sperm capacitation (Guasti et al. Anim.Reprod.Sci. 2017;179:27-34). The present study aimed to stimulate semen motility by adding PTX after thawing of semen previously cryopreserved with different concentrations of CLC. Mangalarga Marchador stallions (n=12; 4-14 years) were assigned to four groups: (G1) untreated control, (G2) inclusion of cholesterol-loaded cyclodextrin (CLC) at concentrations of 1mg, (G3) 1.5mg and (G4) 2mg. Two straws of each stallion per group were thawed and divided into two aliquots, pentoxifylline (2 mg/mL, 7.18 mM) homogenized in BotuCrio®, Botupharma, Brazil (PTX: G1, G2, G3 e G4) was added to one and not to other aliquot (No-PTX: G1, G2, G3 and G4). Sperm kinetics was analyzed by CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®) immediately after thawing (T0) or at 40 minutes (T40), 80 minutes (T80) and 120 minutes (T120) after thawing after incubation at 37°C. For statistical analysis, ANOVA F-test was performed.Intracellular hydrogen peroxide production was estimated by flow cytometry with dichlorodihydrofluorescein diacetate. Fertility was tested by deep horn insemination 0 to 6h after ovulation: No-PTX G1, No-PTX G3 and PTX G3. PTX reduced sperm velocity parameters at all times (p<0.05) compared to the groups without addition of pentoxifylline (No-PTX G1, G2, G3 and G4). Egg yolk based semen extenders may interfere with PTX cell penetration as they reduce permeability of the cell membrane. Regarding the integrity of the sperm plasma membrane and production of hydrogen peroxide, the use of pentoxifylline conferred an increase in sperm possibly under oxidative stress in the control group, with injured plasma membrane and with intracellular hydrogen peroxide (G1 PTX). When PTX was added to the group with highest concentration of cholesterol (G4 PTX), the intactness of the plasma membrane was improved, but with intracellular hydrogen peroxide. Mitochondrial potential and plasma membrane destabilization were also evaluated (n.s.). Pregnancy rates were 50% (2/4) for No-PTX G1, 67% (2/3) for No-PTX G3 and 67% (2/3) for PTX G3. In conclusion, addition of PTX did not interfere with mitochondrial potential, membrane destabilization and conception rate but was detrimental to cell longevity and to sperm kinetics.

Investigation of the effects of syringic acid supplemented to Tris semen diluent on ram semen freezability.

To the best of our knowledge, no research has been conducted to test the effects of syringic acid (SA) on ram semen freezing within the scope of natural antioxidants added to semen extenders. Therefore, this study had two main objectives. First, to test whether adding SA to ram semen freezing extender has a protective effect and contributes positively to sperm kinetic, plasma and acrosome integrity, mitochondrial membrane potential, lipid peroxidation, oxidant and antioxidant and DNA damage parameters after thawing. Second, it was to determine at what concentration the SA supplemented to the extender could be applied by in vitro studies by preserving the fertilization ability of frozen semen at the highest level. In the study, six individuals of Sönmez rams were used. The semen was collected from the rams using an artificial vagina and pooled. The pooled semen was divided into five different groups and extended with 0, 0.5, 1, 2 and 4 mM SA (control C, SA0.5, SA1, SA2 and SA4, respectively). After dilution, the semen samples were kept at 4°C for 3 h, then loaded into 0.25 mL straws and frozen in liquid nitrogen vapour. The SA1 and SA2 groups were higher plasma membrane and acrosome integrity (PMAI), high mitochondrial membrane potential (HMMP), plasma membrane integrity and motility compared to other groups (p < .05). It was observed that SA supplemented to the Tris extender significantly reduced DNA damage, and the lowest values were obtained especially in the SA1 and SA2 treatments (p < .05). Also, lowest MDA level was determined at the SA1 and this was statistically significant compared to SA4 and C (p < .05). In conclusion, it was revealed that SA added to Tris semen extender at 1 and 2 mM treatment doses increased progressive and total motility and preserved PMAI, plasma membrane integrity, HMMP and DNA integrity.

The Effect of Adding Different Levels of Reduced Glutathione to Extender on the Quality of Cooled Ring-Necked Pheasant Semen.

Aim: Artificial propagation of ring-necked pheasant through semen preservation is of significance, as this species is facing enormous threats in its natural habitat. Semen preservation inevitably induces oxidative stress, and exogenous antioxidants need to be investigated for the preservation of ring-necked pheasant semen. Therefore, the current study was conducted to investigate the role of glutathione (GSH) in extender on the liquid storage of ring-necked pheasant semen. Materials and Methods: Semen was collected from 10 sexually mature males, evaluated for sperm motility, and pooled. Pooled semen was aliquoted for dilution with Beltsville poultry semen extender (1:5) at 37°C having GSH levels of 0.0 mM (Control), 0.2, 0.4, 0.6, and 0.8 mM. Extended semen was gradually cooled to 4°C and stored in a refrigerator (4°C) for 48 hours. Semen quality, that is, sperm motility, membrane integrity, viability, acrosomal integrity, and DNA integrity, was assessed at 0, 2, 6, 24, and 48 hours. Results: Sperm motility (%), plasma membrane integrity (%), viability (%), and acrosomal integrity (%) were recorded higher (p < 0.05), whereas DNA fragmentation (%) was recorded lower in extender supplemented with 0.4 mM GSH up to 48 hours of storage compared with 0.2, 0.6, and 0.8 mM GSH concentrations and control. Conclusion: It is concluded that 0.4 mM GSH in extender improves sperm quality parameters of ring-necked pheasant during liquid storage up to 48 hours at 4°C.

Bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage with or without antibiotics in a tropical environment

BackgroundIn tropical environments, boar semen is prepared either from a boar on the same farm as the sow herd or collected in semen collection centers and then transported to other farms. Thus, the semen doses can be used for artificial insemination either immediately or preserved for 2–3 days. The present study investigated the bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage in semen extender with or without antibiotics in Thailand.M&MIn total, 20 Duroc ejaculates were collected. Each ejaculate was diluted in Beltsville Thawing Solution extender either with 0.25 g of gentamicin per liter (ANTIBIOTIC) or without gentamicin (NO-ANITIBIOTIC) to create semen doses containing 3,000 × 106 sperm/100 mL. These were stored at 17 °C for 4 days. Semen characteristics and total bacterial count (CFU per mL, log10) were measured after collection and during storage.ResultsSperm viability was decreased by 6.4% for every 1.0 log10 increase in total bacterial count (p = 0.026) and Staphylococcus spp. were the most frequently isolated across ejaculates. Throughout the 4 days of storage, sperm motility, viability and acrosome integrity in the ANTIBIOTIC group were higher than those in the NO-ANTIBIOTIC group (p < 0.05), while the total bacterial count was lower (1.9 ± 0.1 versus 3.9 ± 0.1 log10, respectively; p < 0.001). Without antibiotic supplementation, the total numbers of bacteria counted on days 2 and 3 of storage were higher than those determined on days 0 and 1 (p < 0.001). Differences in semen quality were detected on days 2 and 3 between the NO-ANTIBIOTIC and ANTIBIOTIC groups in high-viability semen (p < 0.05). However, no differences in sperm quality between the NO-ANTIBIOTIC and ANTIBIOTIC groups were detected in the low-viability semen on each storage day (p > 0.05). On the last day of preservation, Globicatella sanguinis (57.2%), Delftia acidovorans (18.9%) and Micrococcus spp. (5.9%) remained as the top three most abundant contaminants in the semen with antibiotic.ConclusionOur findings contribute new insights toward reducing antibiotics as well as rational antibiotic use in the boar AI industry. The growth of bacteria was significantly greater only after 2 days of preservation in the semen without antibiotic. For semen doses diluted from highly viable ejaculates, it is possible to store for 2 days without any antibiotic supplementation. Moreover, bacterial counts increased at the end of storage in the presence of gentamycin, suggesting the loss of bacteriostatic properties of gentamicin to the growth of bacteria during storage.

Reaction time as a libido indicator and its relation to pre-freeze and post-thaw sperm quality in bulls.

Libido and sperm quality output relationship is already not clear in farm animals. The present study compared reaction time (RT) as a libido indicator and the pre-freeze and post-thaw sperm quality of AI bulls. Before the collection of ejaculates (n = 53, from 22 AI bulls [4.2 ± 1 years of age]), RTs were collected using a chronometer as the interval between the bull's arrival at the semen collection area and his first false mount (FM) on another male. The ejaculates were examined for their volume, concentration and motility. Subsequently, all aliquots were diluted with a commercial semen extender and equilibrated for 3 h before freezing. Frozen semen samples were thawed and examined for sperm kinematics using CASA, plasma membrane and acrosome integrity of sperm (PMAI) by flow cytometry. Additionally, the temperature humidity index (THI) values were assessed during the study. Multiple linear regression analysis was used to analyse the data. The results indicated that THI had a significant effect on libido (p < .001). However, libido had no effect on either pre- or post-thaw sperm quality parameters except for the velocity of the average pathway (VAP) (p < .05). Therefore, relying solely on RT -libido- as an indicator of bull sperm quality at AI stations may not be reliable, as it is a complex behavioural assessment.

Viability of Spermatozoa in Goat's Liquid Semen after Adding Sweet Orange Essential Oil to The Andromed Extender

Aims: To find out the percentage of live spermatozoa after diluting the goat's liquid semen using an andromed extender plus sweet orange essential oil.&#x0D; Study Design: Randomized Block Design.&#x0D; Place and Duration of Study: Sample: Galang, Deli Serdang Regency, Indonesia, between December 2022 and January 2023.&#x0D; Methodology: The research procedure starts with the preparation of semen extender, collection of fresh semen, dilution of semen and equilibration. This research obtained conducted using a Randomized Block Design consisting of 5 treatment levels and five replications. Semen storage using 3 Bucks, which has done for three days. As a treatment is the addition of sweet orange essential oil as much as (P0) 0%, (P1) 0.25%, (P2) 0.5%, (P3) 0.75% and (P4) 1% on the andromed extender. The observed variable was percentage viability spermatozoa evaluated before equilibration and after equilibration.&#x0D; Results: The results showed that the addition of sweet orange essential oil had a not significant effect (P &lt;0.01) to viability of spermatozoa. The results of adding sweet orange essential oil to the extender (after equilibration) were 80% (P0), 80% (P1), 80% (P2), 80% (P3) and 81% (P4).&#x0D; Conclusion: Adding sweet orange essential oil to andromed had no effect on the viability of goat spermatozoa in liquid semen.

Use of the flavonoid taxifolin for sperm cryopreservation from the threatened Bermeya goat breed

Taxifolin is a plant flavonoid effective as an antioxidant. This study aimed to assess the effect of adding taxifolin to the semen extender during the cooling period before freezing on the overall post-thawing sperm variables of Bermeya goats. In the first experiment, a dose-response experiment was performed with four experimental groups: Control, 10, 50, and 100 μg/ml of taxifolin using semen from 8 Bermeya males. In the second experiment, semen from 7 Bermeya bucks was collected and extended at 20 °C using a Tris–citric acid–glucose medium supplemented with different concentrations of taxifolin and glutathione (GSH): control, 5 μM taxifolin, 1 mM GSH, and both antioxidants. In both experiments, two straws per buck were thawed in a water bath (37 °C, 30 s), pooled, and incubated at 38 °C. Motility (CASA) was assessed at 0, 2, and 5 h, and sperm physiology was assessed at 0 and 5 h by flow cytometry (viability, intact acrosome membrane, mitochondria membrane potential, capacitation, intracellular reactive oxygen species —ROS—, mitochondrial superoxide, and chromatin status). In experiment 2, an artificial insemination trial (AI) was included with 29 goats for testing the taxifolin 5-μM treatment on fertility. Data were analyzed with the R statistical environment using linear mixed-effects models. In experiment 1 and compared to the control, T10 increased progressive motility (P < 0.001) but taxifolin decreased total and progressive motility at higher concentrations (P < 0.001), both post-thawing and after the incubation. Viability decreased post-thawing in the three concentrations (P < 0.001). Cytoplasmic ROS decreased at 0 and 5 h at T10 (P = 0.049), and all doses decreased mitochondrial superoxide post-thawing (P = 0.024). In experiment 2, 5 μM taxifolin or 1 mM GSH (alone or combined) increased total and progressive motility vs. the control (P < 0.01), and taxifolin increased kinematic parameters such as VCL, ALH, and DNC (P < 0.05). Viability was not affected by taxifolin in this experiment. Both antioxidants did not significantly affect other sperm physiology parameters. The incubation significantly affected all the parameters (P < 0.004), overall decreasing sperm quality. Fertility after artificial insemination with doses supplemented with 5 μM taxifolin was 76.9% (10/13), not significantly different from the control group (69.2%, 9/13). In conclusion, taxifolin showed a lack of toxicity in the low micromolar range and could benefit goat semen cryopreservation.

Reducing oxidative stress by κ-carrageenan and C60HyFn: The post-thaw quality and antioxidant status of Azari water buffalo bull semen

Azeri water buffalo is a species of great interest due to the high quality of its products such as milk. Due to the decreasing trend of its number and risk of extinction in the future, our attention is directed towards ensuring the preservation of its genetic reserves by keeping its sperm. Using antioxidants in semen extender is one of the ways to reduce the detrimental effects of freezing process on post-thawed quality of spermatozoa. This study was conducted to determine the effect of κ-carrageenan (k-CRG) and C60HyFn supplemented semen extender on the quality of post-thawed Azari water buffalo spermatozoa. A total of 30 semen samples were obtained from three buffaloes using an artificial vagina (twice a week for five weeks = 10 replicates). The samples (n = 3) from each replicate were pooled and divided into equal aliquots to prepare 14 extender groups, including control (C), k-0.2, K-0.4, K-0.6, K-0.8 (containing 0.2, 0.4, 0.8 mg K-CRG/mL, respectively), C-0.1, C-0.2, C-0.4, C-0.8, C-1, C-5, C-10, C-20, and C-40 (containing 0.1, 0.2, 0.4, 0.6, 0.8, 1, 5, 10, 20, 40 μM C60HyFn, respectively), and then frozen. After thawing, motility and velocity parameters, plasma membrane integrity (PMI) and functionality (PMF), DNA damage, Hypo-osmotic swelling (HOS) test, malondialdehyde (MDA), total antioxidant capacity (TAC), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase glutathione activities and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging were evaluated. In vivo fertility was compared between k-0.6, C-1 and control groups. 60 buffalo were inseminated 24 h after the onset of estrus. The diagnosis of pregnancy was performed rectally at least 60 days after fertilization. Total and progressive motility and velocity parameters were improved by k-0.4, k-0.6, k-0.8, C-0.4, C-0.8, C-1, C-5, and C-10 groups) compared to the other groups. Plasma membranes integrity and PMF were improved by k-0.4, k-0.6, C-0.4, C-0.8, C-1, C-5, and C-10 groups compared to other groups, while in terms of sperm DNA damage K-0.4, K-0.6, K-0.8, C-0.2, C-0.4, C-0.8, C-1, C-5, and C-10 groups showed better results compared to the control group. The evidence also showed that k- 0.4, k-0.6, k-0.8, C-0.4, C-0.8, C-1, C-5, and C-10 groups could improve TAC, and decrease MDA levels. Also, k-0.4, k-0.6, k-0.8, C-0.2, C-0.4, C-0.8, C-1, C-5, and C-10 groups could improve GPx, CAT, and GSH levels, but no significant difference was found regarding SOD compared to the other groups. DPPH scavengers were tested by K-0.6, K-0.8 and C-1, C-5, C-10, C-0.8, C-0.4 and C-0.2 groups and compared to other groups improved. The fertility rate [70% (14/20)] was higher in C-1 than other groups. To conclude that k-CRG and C60HyFn supplementation can increase the quality parameters of cryopreserved buffalo semen after thawing and that 1 M C60HyFn can increase in vivo fertility of buffalo semen.

Improvement of rooster semen freezability and fertility rate after sericin supplementation in freezing semen extender.

Semen cryopreservation result in decreased sperm parameters and fertilization ability. Sericin exhibits antioxidant activity by reducing lipid peroxidation resulting from free radicals, which can potentially improve cryopreservation outcomes. The present study aimed to examine the efficacy of various sericin concentrations supplemented with a rooster semen-freezing extender on post-thaw semen quality and fertilizing ability of sperm after cryopreservation. Semen samples were collected from 40 roosters (5 reps), then were pooled, and divided into four groups by the levels of sericin supplementation (0%, 0.25%, 0.50%, and 0.75%) in a freezing extender. Semen suspensions were loaded in medium straw (0.5 mL) and cryopreserved with the traditional liquid nitrogen vapor method. Post-thawed semen was evaluated for sperm motility, sperm viability, and lipid peroxidation. Also, the fertility test was determined. The results showed that supplementation of the freezing extender with 0.50% to 0.75% sericin resulted in greater total motility and progressive motility and lower malondialdehyde levels than the other groups after cryopreservation (p<0.05). However, the viability of 0.75% decreased compared with the value of 0.50% sericin supplementation (p<0.05). Moreover, the fertility and hatchability of total eggs were significantly higher in the 0.50% sericin group than in the other groups (p<0.05). In conclusion, 0.50% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved rooster semen.

The Quality of KUB Rooster Sperm with Vitamin E and C Supplementation During Room Temperature Storage

Antioxidant supplementation to semen extenders can help to maintain rooster sperm quality. This study determined how vitamins E and C affected the quality of KUB sperm at room temperature. Semen collection from 12 KUB roosters used dorso abdominal massage method, twice a week. Semen samples were then diluted using LR-EY extender with three treatments: LR-EY without antioxidant as the control, LR-EY supplemented 0.05 mg/ml vitamin E, and 0.2 mg/ml vitamin C. The diluted semen samples were then stored at room temperature, then motility and viability were examined at 0, 1st, 2nd, and 4th hour. The results showed that the percentage of motility between treatment groups and time points differed not significantly (P>0.05). However, vitamin E supplementation had a significant (P<0,05) effect on the percentage of viability in the 2nd and 4th hour. Supplementation of vitamin E 0.05 mg/ml was able to maintain the viability of KUB rooster sperm at 2nd hour (88.75 ± 1.50%) and 4th hour (84.75 ± 2.63%). It can be concluded that vitamin C cannot maintain sperm quality significantly, but vitamin E can preserve the viability of KUB rooster sperm significantly at room temperature.

Effect of different concentrations of green tea on Cryopreservation of German Shepherd dog

To investigate the Effect of adding different concentrations of green tea extract as an antioxidant on specific physical semen characteristics of German Shepherd dogs during various steps of frozen semen processing this study was carried out. A total (of 20) good Ejaculate semen samples were collected in a plastic bag from German Shepherd dogs aged 3-4 yearsIn different locations (dog breeding).To achieve the current study's main objectives, two experiments were designed through periods extended from the beginning of December 2021 until the end of May 2022. The investigations included studying the physical properties of raw Semen (ejaculate volume, semen color, consistency, motility percentage, progressive motility percentage, dead sperm percentage, sperm abnormalities percentage, and hypo-osmotic swelling test (HOST). After that, semen samples were diluted with Tris supplemented with antioxidants. The physical properties of Semen were followed after cooling and after freezing (post-thawing). In the experiment, ten ejaculates were collected. Dilution of Semen was performed with Tris supplemented with two T1, T2, and T3) were cooled slowly up to 5C and equilibrated, packed into 0.5 ml straws, and put in vapor nitrogen (-196C) for 5-10 minutes after that, stored for 48and 72 hr in liquid Nitrogen (-196C). Progressive motility percentage, dead sperm percentage, sperm abnormalities percentage, and hypo-osmotic swelling test (HOST) were evaluated after cooling and post-thawing (Submerging the straws in the water bath at 37C for 30 seconds). It can be concluded from the present study that adding green tea extract as an antioxidant to the semen extender improves the ability to freeze, especially in the treatment T3 (0.01 mg/5 ml) and the T2 (0.05 mg/5 ml). This additive can decrease the negative Effect of freezing in liquid Nitrogen compared to the control group.

Antimicrobial activity of cell free supernatants from probiotics inhibits against pathogenic bacteria isolated from fresh boar semen

The use of antibiotics with semen extender appears to be a practical solution to minimise bacterial growth in fresh boar semen preservation. Unfortunately, the excessive use of antibiotics promotes antimicrobial resistance (AMR). This becomes a worldwide concern due to the antimicrobial resistance genes transmitted to animals, environment, and humans. Probiotics are one of the alternative methods to reduce antibiotic use. They could inhibit pathogenic bacteria by producing antimicrobial substances in cell free supernatants (CFS). Nevertheless, there is no comprehensive study undertaken on inhibitory activity against pathogenic bacteria isolated from boar semen origin. Our study investigated the efficacy of CFS produced from selected probiotics: Bacillus spp., Enterococcus spp., Weissella spp., Lactobacillus spp., and Pediococcus spp. inhibiting pathogenic bacteria isolated from fresh boar semen. Besides, the semen-origin pathogenic bacteria are subjected to identification, antimicrobial resistance genes detection, and antibiotic susceptibility test (AST). Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis are the most common pathogens identified in boar semen with resistance to numerous antibiotics used in pig industry. The CFS with its antimicrobial peptides and/or bacteriocin constituent derived from selected probiotics could inhibit the growth of pathogenic bacteria carrying antimicrobial resistance genes (mcr-3 and int1 genes). The inhibition zones for Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis provided more efficient results in the CFS derived from Lactobacillus spp. and Pediococcus spp. than those of the CFS produced from Enterococcus spp., Weissella spp. and Bacillus spp., respectively. It is worth noted that as the incubation time increased, the antibacterial activity decreased conversely. Our results on CFS with its antimicrobial peptides and/or bacteriocin constituent inhibits semen-origin pathogenic bacteria guide the direction as a promising alternative method used in the semen extender preservation of the pig industry.

Tinospora cordifolia modulates the seminal parameters, leakage of intracellular enzymes and seminal antioxidants in equilibrated and cryopreserved semen of Sahiwal bulls.

The present study was undertaken to assess the effects of stem extract of Tinospora cordifolia (Giloy or Guduchi) in the semen extender on seminal parameters, leakage of intracellular enzymes and antioxidants in semen of Sahiwal bull. A total of 48 ejaculates from four bulls were selected for the study. Spermatozoa of 25×106 were incubated in 100, 300 and 500 μg of stem extract of Guduchi as Gr II, III and IV, respectively and pre-freeze and post-thaw semen samples were analysed for seminal parameters (motility, viability, total sperm abnormality; TSA, plasma membrane integrity; PMI, and acrosomal integrity; AcI), intracellular enzymes (aspartate aminotransferase; AST and lactate dehydrogenase; LDH) and seminal antioxidants (superoxide dismutase; SOD and catalase) in comparison with an untreated control group (Gr I). The results revealed that stem extract treated semen had significantly (p<0.05) higher motility, viability, PMI, AcI, SOD and catalase and had significantly (p<0.05) lower TSA, AST and LDH compared to those in untreated control group at pre-freeze and post-thaw stages. Semen treated with 100 μg stem extract /25×106 spermatozoa had significantly (p<0.05) higher motility, viability, PMI, AcI, SOD and catalase and had significantly (p<0.05) lower TSA, AST and LDH compared to those in control, 300, and 500 μg treated groups at pre-freeze and post-thaw stages. Further, these seminal parameters and antioxidants were showing decreasing trend and TSA and leakage of intra-cellular enzymes were showing increasing trend from Gr II to Gr IV at pre-freeze and post-thaw stages. Thus, 100 μg/25×106 spermatozoa were optimum or suitable dose for cryopreservation of Sahiwal bull semen. The study concluded that T. cordifolia stem extract 100 μg/25×106 spermatozoa in the semen extender can be effectively utilized to reduce the oxidative stress and improve the pre-freeze and post-thaw seminal parameters in Sahiwal bull. However, further studies on effects of different concentrations of stem extract on in-vitro or in-vivo fertility trials are to be conducted to assess the impact of the stem extract supplementation in the semen extender on field pregnancy outcome in bovine species.

The Effects of Water-Based Coconut Extenders on Semen Preservation : A review

For animal reproduction, artificial insemination has become a need. Semen preservation has been crucial in artificial insemination as it can determine the success of fertilization and pregnancy. Semen extenders have been critical in prolonging the usability of semen while also retaining its viability, motility, and integrity so that it can be used to fertilize female animals. Due to its nutritional and chemical qualities that can create an environment for spermatozoa to survive and thrive before insemination, coconut water has been the focus of various research addressing its effect on semen preservation. These studies have shown that coconut water has the potential to be an effective semen extender due to its ability to protect the semen during cooling and cryopreservation. Coconut water also includes nutrients (water, sugars, proteins, salts, and vitamins) to help spermatozoa thrive in a healthy environment. However, its performance lags that of commercial extenders, but it can be a less expensive alternative or replacement when commercial extenders are unavailable.

Studi Literatur Efektivitas Penggunaan Berbagai Jenis Pengencer yang Ditambahkan Antioksidan Terhadap Kualitas Semen Kambing (Capra aegagrus hircus)

Goats are ruminants that have considerable potential. The livestock population data in the province of East Nusa Tenggara (NTT) has continued to increase from 2004 to 2018. The Artificial Insemination (AI) program has a function to improve genetic quality and livestock production. Extender is a mixture of various materials that can maintain the quality of spermatozoa in animal semen. Antioxidants to prevent the emergence of oxidant compounds in excess. The purpose of this literature study was to determine which semen extender has effectiveness in improving the quality of goat semen and the active substance that acts as an antioxidant as one of the substances that can improve the quality of the extender. The process of this literature study is carried out by searching and collecting various kinds of existing literature using Google Scholar, Google Patent, Pubmed then all existing references are compiled with the Mendeley application. Reference sources obtained are scientific journals, scientific articles/publications, e-books and national and international seminar proceedings by searching for "keywords" the effectiveness of using various types of extenders added with antioxidants on the quality of goat semen collected with a total of approximately 85 references. The results obtained from the literature study carried out are on the effectiveness of the semen extender, namely the more effective commercial extender, namely Andromed®, then the natural extender, namely guava leaves, while the compound extender is TGC+CLC (tris citrate glucose + Cholesterol Loaded). Cyclodextrin). Antioxidant active substances, both commercial and natural, that can increase the quality of goat semen, namely commercial antioxidants are Glutathione + 6 mM, while natural antioxidants are TKT (Egg Yolk Tris) + 4% Carrot Extract. The temperature and storage time of goat semen, the optimal temperature used for the equilibration time temperature is 5℃, at the cryopreservation temperature at -196℃, then at the optimal storage

Ethanolic Purslane (Portulaca oleracea) Leaf Extract Improves the Quality of Cryopreserved Goat Semen

Portulaca oleracea contains a wide range of chemical constituents, including flavonoids, alkaloids, polysaccharides, fatty acids, terpenoids, sterols, proteins, vitamins, and minerals. These chemical constituents are linked to the plant’s diverse pharmacological properties, such as antimicrobial and antioxidant activities. Therefore, this study is aimed at evaluating the effect of adding two concentrations (50 or 100 μg/mL) of Portulaca oleracea leaf ethanolic extracts in a native form (POLE) or nanoformulation (POLENF) on the antioxidant status of sperm medium to improve goat sperm freezability. Semen was evaluated postequilibration (5°C for 2 h), postthawing, and 2 h postincubation (37°C and 5% CO2). Phytochemical analysis showed the presence of 30 compounds in POLE, including phenolic acids and flavonoids and their glucosides. Total flavonoids were higher in POLE than in POLENF. Both extracts showed strong free radical scavenging capacity by the 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay, which was higher for POLENF than POLE, whereas the antioxidant activity by the ferric reducing antioxidant power assay had no differences. In comparison to free extender, the addition of POLE (100 μg) or POLENF (50 and 100 μg) significantly improved sperm motility and viability postequilibration and postincubation and antioxidant status of sperm medium postthawing. All POLE and POLENF concentrations significantly improved motility, viability, membrane integrity, and apoptosis of spermatozoa postthawing. Enzyme activities of AST, ALT, and LDH in sperm medium postthawing were not affected by treatments. In comparing different types and concentrations of POLE with free extender, the addition of POLENF (100 μg/mL) to goat semen extender gave the most valuable results as an effective free radical scavenger and antiapoptotic during cryopreservation.

Freezability and Fertility Rates of Stallion Semen Supplemented With Trehalose in Lactose Extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. One of the reason for this diminished quality is osmotic stress that spermatozoa experiences during freezing and thawing process. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze–thawing process. Semen samples were collected from six Marwari breed stallions, divided into three different treatments in a final concentration of 150 × 106 sperm/mL by using Lactose based extender containing 0, 50, and 150 mM of trehalose then subjected to cryopreservation after equilibration. Sperm motility, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, DNA integrity and oxidative stress related parameters of the stallion spermatozoa were analyzed at fresh, prefreeze and post thaw stages. Thirty (30) reproductively healthy mares were inseminated with frozen-thawed semen either supplemented with (treatment) or without (control) trehalose to evaluate the field fertility. Results of the current study indicated that, the extender containing 50 mM trehalose has enhanced the functional plasma membrane, acrosomal, DNA integrities and augmented the mitochondrial membrane potential. Trehalose supplementation to the semen extender not only ameliorated the semen quality parameters, but also protected the stallion sperm from oxidative stress by reducing the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO). The inclusion of 50 mM trehalose in semen extender resulted in significantly (P < .05) increased post-thaw progressive motility and viability compared to the control group. Mares inseminated with frozen-thawed semen supplemented with 50 mM trehalose tended to have better pregnancy rates than controls (non-significant [P < .05]) although a larger fertility trial is required to determine if this effect reaches the level of significance. In conclusion, addition of 50 mM trehalose yielded in better quality stallion semen after cooling and post-thawing in terms of reducing the oxidative stress and enhancing the motility, integrities of acrosome, plasma membrane, mitochondrial potential and DNA.

Growth Dynamic and Threshold Values for Spermicidal Effects of Multidrug-Resistant Bacteria in Extended Boar Semen.

The aim of this study was first to examine the prevalence of bacteria-associated loss of sperm quality in samples from insemination centers during a seven-year semen monitoring program and, second, to investigate the growth dynamic of four different multidrug-resistant bacterial species and their impact on sperm quality during semen storage. A reduced sperm quality associated with bacterial contamination was found in 0.5% of 3219 of the samples from insemination centers. In samples spiked with Serratia marcescens and Klebsiella oxytoca, bacterial growth by six log levels was seen during storage at 17 °C, causing loss of sperm motility, membrane integrity, membrane fluidity, and mitochondrial membrane potential at >107 CFU/mL (p < 0.05). Storage at 5 °C in the Androstar Premium extender efficiently inhibited their growth. Achromobacter xylosoxidans and Burkholderia cepacia showed limited growth up to two log levels at 17 °C and did not impair sperm quality. In conclusion, spermatozoa tolerate moderate loads of multidrug-resistant bacteria, and hypothermic, antibiotic-free semen storage effectively limits bacterial growth. The constant use of antibiotics in semen extenders should be reconsidered.