Extenders For Cryopreservation Research Articles

Effects of Supplementation of Different Antioxidants to Cryopreservation Extender on the Post-Thaw Quality of Rooster Semen-A Meta-Analysis.

The standardization of the semen cryopreservation technique could be an effective tool in poultry for the conservation of genetic resources. During this process, the production of reactive oxygen species increases, leading to oxidative stress that causes damage to the spermatozoa. To reduce this effect, the addition of exogenous antioxidants in the cryopreservation diluent has been reported to be effective. Multiple antioxidants such as catalase, vitamin E, cysteamine, ergothioneine, and serine have been studied in roosters. Therefore, the present investigation aims to perform a meta-analysis to determine if the use of the aforementioned antioxidants added to the cryopreservation extender produces an improvement in semen quality parameters in roosters after thawing. After collecting the data, a discriminant canonical analysis was performed to determine which of the selected semen quality traits provided the most information, with hypo-osmotic swelling test (HOST), viability, and total motility variables showing the highest discriminatory power. However, according to the descriptive statistics, catalase and serine are the antioxidants that improve a greater number of seminal quality parameters, and since catalase gives the most favorable results for most of the discriminating variables, it could be the antioxidant of choice.

Effect of Supplementation of a Cryopreservation Extender with Pectoliv30 on Post-Thawing Semen Quality Parameters in Rooster Species.

Sperm cryopreservation is a fundamental tool for the conservation of avian genetic resources; however, avian spermatozoa are susceptible to this process. To cope with the high production of reactive oxygen species (ROS), the addition of exogenous antioxidants is beneficial. Pectoliv30 is a substance derived from alperujo, and in this study, its effect was analyzed on seminal quality after its addition to the cryopreservation extender of roosters at different concentrations. For this purpose, 16 Utrerana breed roosters were used, and seminal collection was performed in six replicates, creating a pool for each working day with ejaculates of quality. After cryopreservation, one sample per treatment and replicate was thawed, and several seminal quality parameters were evaluated. Statistical analysis revealed numerous correlations between these variables, both positive and negative according to the correlation matrix obtained. Furthermore, the chi-squared automatic interaction detection (CHAID) decision tree (DT) reported significant differences in the hypo-osmotic swelling test (HOST) variable between groups. Moreover, results for this parameter were more desirable at high concentrations of Pectoliv30. The application of this substance extracted from the by-product alperujo as an antioxidant allows the improvement of the post-thawing seminal quality in roosters and facilitates optimization of the cryopreservation process as a way to improve the conservation programs of different endangered poultry breeds.

Application of antioxidants in extender on bull sperm cryopreservation to reduce the male effect in dairy fertility

Because male and female effects on fertility must be considered, it may be difficult to achieve accurate and repeatable fertility predictions using only sperm characteristics given differences in breed, health, and season. Improving sperm quality after cryopreservation may be a method to reduce the male effect on the fertility outcome. This study was conducted using 2 different Certified Semen Service approved extenders, one containing plant-derived antioxidants, to assess cryopreserved sperm quality and determine pregnancy per AI (P/AI) in a commercial dairy farm. Beef bull ejaculates (n = 24 ejaculates, n = 4 bulls) were split and frozen in egg-yolk-TRIS (EYT) or GameteGuard-FB cryopreservation extenders. Frozen-thawed sperm was evaluated for sperm quality and used for insemination in dairy cows (n = 4,856). Improvement was shown in total and progressive motility, acrosome integrity, and reduction of DNA fragmentation at 0 and 3 h after thawing in sperm frozen in GameteGuard-FB when compared with the EYT extender. An overall 12.7% improvement in pregnancy per AI (P/AI) was observed when GameteGuard-FB (40.8%) was used for cryopreservation when compared with EYT (36.2%). More importantly, GameteGuard-FB frozen semen increased P/AI for first service by 19.5% (35.9% EYT and 42.9% GameteGuard-FB) and 45.0% was also observed at third service (31.3% EYT and 45.4% GameteGuard-FB). The use of GameteGuard-FB resulted in an average 40% P/AI among bulls. Taken together, the increase in cryopreserved sperm quality using GameteGuard-FB can be used to minimize the male effects on pregnancy outcomes in commercial dairy farms.

Impact of low-dose ozone supplementation on motility parameters and bacterial growth in horse cryopreserved semen

Two studies were conducted to evaluate the use of medical ozone (O3) in commercial extenders for equine semen cryopreservation. In the first study (Study 1), 0, 5, and 15 µg/mL of O3 were added to diluents of refrigerated or frozen semen. Samples were evaluated for sperm kinematics at different time points for the chilled samples and after a thermoresistence test for the frozen/thawed samples. In the second study (Study 2), 0, 5, and 10 µg/mL of O3 were added to an antibiotic-free diluent for refrigerated semen for comparison with the control group in which semen was diluted in the same diluent enriched with antibiotics. Semen sample kinematics were analyzed and an aliquot was collected after ozonification for bacteriological analyses. For Study 1 no difference was found comparing all the kinematic parameters analyzed over time, in the various treatments (P > 0.05). In Study 2 the absence of antibiotics did not affect the kinematic parameters compared to the control (P > 0.05). However when antibiotics were added, a smaller number of bacterial colony-forming units were detected compared to samples without antibiotics and without or with different O3 supplementations. In conclusion, O3 treatment at low dosages did not affect the semen kinematics, although it was ineffective in preventing bacterial overgrowth. Higher O3 concentrations should be evaluated to explore the possibility of reducing the use of antibiotics in equine sperm conservation.

Effect of Three Semen Extenders on Sperm Quality and In Vitro Fertilization Rates of Fresh and Cryopreserved Sperm Collected from Llama (Lama glama) Vas Deferens.

The advances in Assisted Reproductive Technologies (ARTs) applied in South American camelid species are still scarce. The aim of this study was to compare the effects of three semen extenders, before and after the cryopreservation of spermatozoa obtained from the vas deferens, on sperm quality parameters and in vitro fertilization rates of llama (Lama glama) oocytes. Mature fertile llama males (Lama glama; n = 6; age: 48-60 mo.; BCS: ~2.7) were included in the study. Sperm samples were collected from each male using the surgical technique of the vas deferens deviation. Then, the sperm samples were pooled and diluted with the Tris-EY, Andromed®, or BioxCell® extender in order to subsequently carry out the sperm cryopreservation process. The sperm quality assessment related to each extender was performed before and after cryopreservation with regard to sperm morphological abnormalities, acrosome integrity, sperm viability, membrane permeability, and sperm motility traits. Moreover, in vitro fertilization (IVF) procedures were carried out to evaluate the in vitro fertility of the cryopreserved sperm samples using each extender. Overall, significant differences were observed before and after cryopreservation regarding acrosome integrity, sperm viability, membrane permeability, and sperm motility traits among the extenders used, where Tris-EY and Andromed® were better than BioxCell® (p < 0.05); however, no differences were observed regarding the sperm morphological abnormalities among extenders (p > 0.05). Moreover, multiple differences were observed with regard to the velocity and linearity kinematic parameters obtained by computerized analysis before and after the cryopreservation process, irrespective of the extender used (p < 0.05). Finally, differences were observed regarding the in vitro fertilization rates among the different extender-derived samples (p < 0.05). In conclusion, the sperm quality using Tris-EY and Andromed® was better before and after cryopreservation compared to that using BioxCell®. Although the number of fertilized oocytes obtained after the IVF process between Tris-EY and Andromed® was similar, Andromed®-derived samples showed the best sperm quality results before and after cryopreservation. This indicates that the cryopreservation extender is a determining factor in significantly improving in vitro fertilization rates when using sperm samples obtained from vas deferens in llama (Lama glama) males.

Evaluation of equine semen frozen in extenders free of egg yolk using two different freezing curves

A chemically defined cryopreservation extender that maintains seminal parameters is relevant. Fifteen ejaculates from 5 stallions (n= 5; r=3) were diluted in 5 extenders: 1) EDTA-glucose based extender with egg-yolk and dimethylformamide (EY); 2) commercial equine extender (CE); 3) CE with dimethylformamide (CE-3); 4) bovine commercial extender with liposomes (OP); 5) bovine commercial extender with soybean lecithin (BIO), and frozen using a slow and a rapid temperature descent curve. Post-thaw evaluations were: sperm kinematic parameters, viability and acrosome status, membrane lipoperoxidation and DNA fragmentation. Sperm data were analysed using an ANOVA or Friedman test (results mean ± SD). Paired comparison between the two freezing curves was analysed using the Wilcoxon test. Total and progressive motility were significantly higher (P<0.05) in the EY and CE-3 samples using the slow curve, whereas for the fast curve, total and progressive motility were significantly higher (P<0.05) in the EY samples compared to all the extenders and the samples frozen in CE-3 were significantly higher than the remaining extenders (P<0.05). The percentages of live acrosome intact sperm and of live non-peroxidized sperm were significantly higher (P<0.05) in the EY extender when using either of the freezing curves and in turn, were significantly higher (P<0.05) in samples frozen in CE-3 compared to the remaining extenders. Intact DNA was significantly lower (P<0.05) in the BIO extender, using the rapid curve. To conclude, the commercial equine extender with 3% dimethylformamide, without egg-yolk, could be a suitable alternative for extenders with egg-yolk.

Evaluation of the effect of the addition of an olive oil-derived antioxidant (Pectoliv-80A) in the extender for cryopreservation of rooster sperm through the use of a discriminant statistical tool

During the poultry sperm cryopreservation process, an excess of reactive oxygen species is generated resulting in oxidative stress which harms the quality of avian spermatozoa. To counteract this effect, the addition of exogenous antioxidants, such as Pectoliv-80A (a by-product of olive oil), to the cryopreservation diluent is interesting. For this purpose, 16 roosters belonging to the Utrerana avian breed were used. Six semen pools (from the 6 different replicates) were divided into 4 aliquots corresponding to different concentrations of Pectoliv-80A that were tested (0, 300, 400, and 500 μg/mL), and the cryopreservation process was carried out. To evaluate post-thawing semen quality, different parameters such as motility, membrane functionality, reactive oxygen species production, lipid peroxidation, and acrosome integrity were studied. A discriminant canonical analysis was used to determine both the differences between the Pectoliv-80A concentration groups and the discriminant power of the aforementioned parameter used for semen evaluation. Total motility and membrane functionality were reported to be the most discriminant variables for differentiating the different antioxidant enrichment groups and concluded that concentrations of 300 μg/mL showed the most desirable quality of post-thawing semen. The present study could lead to the optimization of both cryopreservation and quality evaluation techniques of the sperm of rooster species, that support the conservation program of endangered local breeds.

Evaluation of Cryopreserved Ram Sperm with Nano-Ozone Solution and Post-Thaw Life Span by Flow Cytometric Analysis.

Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 μg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 μg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) (p < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.

Addition of low concentration of cholesterol-loaded cyclodextrin (CLC) has a positive effect on cryopreserved canine spermatozoa evaluated by andrological and biophysical methods

BackgroundThis study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP).ResultsAn addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05).ConclusionsIn conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters.

Preservation of rooster post-thawed sperm epigenetic modifications, fertility potential and other quality parameters in different extenders using reduced glutathione

Although rooster semen cryopreservation is an efficient procedure to spread qualified semen samples for reproductive goals, some post-thawed qualified semen samples resulted in poor fertility rate that could be related to epigenetic modifications during the cryopreservation process. This research was conducted to investigate the effect of reduced glutathione (GSH) in different cryopreservation extenders (Lake and Beltsville) on preservation of epigenetic modifications, fertility potential and other quality parameters of rooster sperm after thawing. Semen samples were collected and diluted in Lake and Beltsville extenders as follows: L-0: Lake without GSH, L-G: Lake with GSH, B-0: Beltsville without GSH, and B-G: Beltsville with GSH. After freeze-thawing process, sperm motility, membrane functionality, mitochondrial activity, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation, ROS concentration, epigenetic modifications and fertility potential were evaluated. In results, the type of extender had no effect (P > 0.05) of post-thawed sperm quality. The treatments containing GSH presented higher (P ≤ 0.05) total motility, progressive motility, membrane functionality, mitochondrial activity, acrosome integrity, viability, DNA methylation, fertility as well as lower (P ≤ 0.05) lipid peroxidation, apoptosis, DNA fragmentation and ROS concentration than other treatments. Extender supplementation with GSH had no effect (P > 0.05) on histone methylation, histone acetylation and hatching rate. In conclusion, supplementation of rooster sperm cryopreservation extender with GSH could be an effective strategy to preserve post-thawed sperm DNA methylation, fertility and other quality parameters during reproductive programs.

In Vitro and In Vivo Effect of Oxytetracycline on Sperm Parameters In Breeding Rooster

BACKGROUND: Some antimicrobials could adversely affect sperm quality during sperm cryopreservation and antibiotic treatment with subsequent effects on fertility outputs. To our knowledge, no similar studies have been conducted on breeding roosters, especially for oxytetracycline (OTC). OBJECTIVE: To investigate both in vitro and in vivo impact of oxytetracycline on sperm parameters in breeding roosters. METHODS: Sperm motility parameters were objectively analyzed using the CASA system including total motility (TM %), progressive motility (PM %), all sperm velocities, the sperm count, and cell viability during 9 days of in vivo treatment. In the in vitro investigation, the pooled sperm was diluted and divided into a control aliquot (diluted in 0.9% NaCl) and treated samples. Motility parameters were assessed after 0, 1, 2, 3, 4, 5, and 6 hours of storage at 37ºC. In the in vivo study, 1 g/L of OTC was administrated to five individuals for nine consecutive days. Fresh semen samples were analyzed at T0 (before treatment) and after 6 (T6) and 9 days (T9) of treatment. RESULTS: OTC caused significant impairment of sperm quality in vivo. A drastic reduction in sperm concentration, viability, TM, PM, and all kinematic parameters was observed after 6 days of treatment. However, at day 9 sperm quality had improved to be nearly similar to T0. In vitro, OTC induced similar sperm impairment on all sperm motility parameters. CONCLUSION: Oxytetracycline exhibited negative effects on rooster sperm both in vivo and in vitro and appears consequently not suitable in cryopreservation extenders.

Proteomic analysis of high and low-motility frozen-thawed spermatozoa in yak provides important insights into the molecular mechanisms underlying sperm cryodamage

Sperm cryodamage caused by cryopreservation limits the use of frozen yak spermatozoa in artificial insemination (AI). However, the proteomic changes involved in the cryodamage of yak spermatozoa have not been investigated to date. Therefore, this study aimed to identify proteins related to freezing tolerance. Tandem mass tag (TMT) were used in combination with liquid chromatography–tandem mass spectrometry (LC-MS/MS) for identifying differentially expressed proteins (DEPs) between high-motility (HM) and low-motility (LM) frozen-thawed yak spermatozoa. A total of 116 DEPs were identified (>1.5-fold, P < 0.05); of which, 104 proteins were upregulated in HM spermatozoa and 12 proteins were upregulated in LM spermatozoa. The results of functional annotation analysis revealed that the DEPs were mainly involved in metabolic processes. A total of 20 DEPs that were abundantly expressed in HM spermatozoa were strongly associated with carbohydrate metabolism. The results of KEGG analysis revealed that the DEPs were enriched in glycolysis/gluconeogenesis, PPAR signaling pathway, and Ras signaling pathway. In addition, many antioxidant enzymes such as superoxide dismutase (SOD1), peroxiredoxin-6 (PRDX6), and Parkinson disease protein 7 (PARK7) were upregulated in HM spermatozoa, suggesting that these enzymes affect the motility of spermatozoa by regulating the levels of reactive oxygen species (ROS) in frozen-thawed spermatozoa. Altogether, the findings of this study elucidate the mechanisms through which cryopreservation affects the movement of yak spermatozoa and offer a novel basis for refining freezing techniques and modifying cryopreservation extender components.

Effect of Honey, Coenzyme Q10, and β-Carotene/α-Tocopherol as Novel Additives in Rabbit-Sperm Cryopreservation Extender.

The effectiveness of rabbit-sperm cryopreservation is still below average compared to other domestic species. After the sperm cryopreservation process, post-thawing parameters like motility and membrane integrity are significantly compromised. The use of new extender constituents is an approach that can be used to improve the effectiveness of cryopreservation. Accordingly, we used honey (1.25, 2.5, 5, and 10%), coenzyme Q10 (100 and 200 μM), and β-carotene/α-tocopherol (500 μM/620 μM and 250 μM/310 μM) as candidate components for rabbit-sperm extenders during cryopreservation. Ejaculates from commercial adult rabbit bucks (n = 5) were cryopreserved using conventional freezing. Several post-thawing sperm parameters were assessed, including total motility, membrane integrity, viability, nuclear membrane integrity, acrosome reaction, and mitochondrial membrane potential and activation. Additionally, we performed hormonal analyses of the seminal plasma. Moreover, we analyzed the post-thawing levels of a molecular marker of sperm quality, proAKAP4, which was used in rabbits for the first time. Our findings showed that the 2.5% honey supplementation increased the post-thawing sperm motility (13.75 ± 3.75%) compared to the greater concentrations employed. However, the post-thawing motility was negatively affected by the coenzyme Q10 (0%, in both groups) but was not affected by the β-carotene/α-tocopherol supplementation (22 ± 18.15%, and 11.67 ± 10.17%). In conclusion, the cryopreservation protocols of this study did not help to maintain the sperm parameters after thawing. Further studies are required to identify novel protocols to mitigate the damage caused to rabbit sperm during cryopreservation.

Influence of Equilibration Time and Bull-Specific Extender for Cryopreservation on Semen Quality and Fertility in German Holstein Friesian Bulls: A Controlled Field Trial.

Equilibration with an extender is necessary to allow cryopreservation of bovine sperm. The aim of trial 1 was to assess the effect of 24 h versus 4 h equilibration time with three different extenders on sperm quality and to select the preferred extender for each bull. The aim of trial 2 was to investigate the effect of using a 24 h equilibration time with a bull-specific extender on field fertility. For trial 1, three ejaculates each from eight Holstein Friesian breeding bulls were used as the split-sample, including two equilibration times (4 h and 24 h) and three extenders (BioXcell, Triladyl, and OptiXcell). For trial 2, from 5 to 10 ejaculates from the same bulls were collected and treated (split-sample) as BioXcell with 4 h equilibration and either Triladyl or OptiXcell, both with 24 h equilibration. A total of 11,059 straws were used for insemination of cows and heifers. For Triladyl, progressive sperm motility, acrosome defects, and plasma membrane and acrosome integrity improved with a 24 h compared to a 4 h equilibration time. Four bulls each were used with Triladyl and OptiXcell for trial 2. In trial 2, non-return rates did not differ among groups. Therefore, using a 24 h equilibration time might improve in vitro sperm parameters, depending on the extender used. Moreover, it would be possible to change from 4 h to 24 h equilibration time without impairing field fertility.

Enhancing post-thaw quality of ram epididymal sperm by supplementation of rutin in cryopreservation extender

The purpose of this study was to examine the effect of different rutin concentrations on rams epididymal sperm. A local slaughterhouse provided 50 pair of testes from 25 rams. The testes were sent to the lab at room temperature. Spermatozoa were extracted by suspending portions of cauda epididymis in tris solution. Ram sperm was cryopreserved (in liquid nitrogen) in a tris extender containing rutin at 0, 0.5, 0.75, 1, and 1.25 mM. Rutin showed superior sperm total and progressive motility, beat cross frequency, straight line velocity, velocity average pathway and membrane integrity values at 0.75 and 1 mM. The morphology of the sperm and the superoxide dismutase levels did not significantly change with different treatments. Moreover, rutin at 0.75 and 1 mM was also shown to have the highest level of mitochondrial activity. The results showed ATP, total antioxidant capacity, and glutathione peroxidase levels were significantly greater in the rutin 0.75 and 1 mM groups (P < 0.05). Rutin at 0.75 and 1 mM levels had the lowest reactive oxygen species concentrations. Rutin at 0.75 and 1 mM substantially increased the proportion of viable sperm (P < 0.05). The lowest amount of apoptosis was observed in 0.75 and 1 mM rutin. Rutin at 0.75 and 1 mM yielded the least significant percentage of dead sperm. It may be inferred that adding 0.75 and 1 mM to the sperm extender can enhance the quality of the epididymal sperm in rams after the cryopreservation process.

Phosphocreatine addition to extender enhances the quality and antioxidant capacity of cryopreserved boar sperm.

Boar sperm are less resistant to drastic changes in the external environment during cryopreservation, mainly because their plasma membranes are rich in unsaturated fatty acids but lack cholesterol and are thus susceptible to lipid peroxidation caused by the attack of reactive oxygen species. This study evaluated the effect of adding phosphocreatine to cryopreservation extenders on boar sperm quality and antioxidant capacity. Different concentrations (0, 5.0, 7.5, 10.0 and 12.5 mmol/L) of phosphocreatine were added to the cryopreservation extender. After thawing, sperm were analysed for morphological parameters, kinetic parameters, acrosome integrity, membrane integrity, mitochondrial activity, DNA integrity and antioxidant enzyme activity. The results showed that 10.0 mmol/L phosphocreatine samples enhanced the boar sperm motility, viability, average path velocity, straight-line velocity, curvilinear velocity and beat cross frequency after cryopreservation and reduced the malformation rate compared to the control group (p < .05). The acrosome integrity, membrane integrity, mitochondrial activity and DNA integrity of boar sperm were higher than those of the control group after adding 10.0 mmol/L phosphocreatine to the cryopreservation extender (p < .05). Extenders containing 10.0 mmol/L phosphocreatine maintained high total antioxidant capacity; elevated the activities of catalase, glutathione peroxidase and superoxide dismutase; reduced malondialdehyde and H2 O2 content (p < .05). Therefore, adding phosphocreatine to the extender is potentially beneficial for boar sperm cryopreservation at an optimal 10.0 mmol/L concentration.

Epsilon poly-lysine in buffalo semen extender: A step towards reducing the development of antibiotic resistance.

The use of antibiotics in semen extenders can contribute to the development of antibiotic resistance. The objective of the study was to evaluate epsilon-polylysine (Ɛ-PL) as a substitute for antibiotics in the buffalo semen extender. For this, 20 semen ejaculates were collected from four Murrah buffalo bulls. Each ejaculate was divided into three equal aliquots and extended into an egg yolk-based semen extender containing either antibiotics (strepto-penicillin) or different concentrations of Ɛ-PL (0.64 and 1.28 g/L) to make the final concentration 80 million sperm/mL and cryopreserved as per the standard procedure. The antibiogram sensitivity test confirmed that Ɛ-PL is an effective antimicrobial against microbes present in buffalo semen ejaculates. Furthermore, the addition of Ɛ-PL in the semen extender significantly reduces the colony forming unit (CFU)/mL in cryopreserved semen equivalent to strepto-penicillin. The sperm motility and kinematic parameters assessed by a computer-assisted sperm analyser showed that Ɛ-PL did not inhibit either sperm motility not kinematic parameters of cryopreserved sperm. The flow-cytometric evaluation of frozen-thawed sperm revealed interesting results. The extender supplemented with Ɛ-PL protected sperm acrosome and mitochondrial membrane potential greater than the extender supplemented with strepto-penicillin. Further, Ɛ-PL reduced significantly the production of superoxide anions from mitochondria during the cryopreservation process. In this way, Ɛ-PL may be a suitable alternative to antibiotics in semen extenders. In conclusion, Ɛ-PL at a concentration of 0.64 g/L acts as an effective antimicrobial as well as antioxidant in semen extender for cryopreservation of buffalo sperm.

Endometrial inflammatory response to insemination in jennies

In the jenny introduction of semen into the uterus triggers an endometrial inflammatory response characterized by polymorphonuclear (PMN) infiltration. The presence of cryoprotective agents in frozen-thawed semen may increase the inflammatory reaction. The objective of this study was to evaluate the effects of two cryoprotective agents in the induction of endometrial inflammatory response in jennies inseminated with extended semen or sham-inseminated with extender only or with deionized water. Eight Creole donkeys were inseminated or sham-inseminated in successive cycles with each one of the following treatments: DW: deionized water; B: a commercial cryopreservation extender containing glycerol (BotuCrio®, Botupharma); LDL: a cryopreservation extender that contains low density lipoproteins instead of glycerol (UNAM-extender); FS: fresh semen in a extender which contains no cryoprotective agents (INRA96, France); S-B: Semen extended in Botucrio; and S-LDL: Semen extended in UNAM-extender. The order in which jennies received each treatment was randomized. In each case, four 0.5 ml straws were used for deep insemination in the uterine horn ipsilateral to ovulation. In the case of treatments that included semen, each straw contained 200 million sperm cells. After each treatment the jennies were administered 5 mg of PGF2α at day 5 post-ovulation to induce return to estrus in order to administer the next treatment, until each jenny had received allthe treatments. Endometrial cytology samples were obtained before each treatment (time 0) and at 8, 12, 24 and 48-h after treatment. Smears were fixed and stained with Diff-Quik. Five fields were randomly chosen for microscopic observation. The total number of PMN and the total number of cells of any type were counted. The proportions of PMN in the smears were compared using the "generalized linear models" procedure of the SPSS program. Predictors were treatment, post-treatment time, treatment x time interaction. The jenny was used as a block. For multiple comparisons, the Bonferroni method was used. Extenders with cryoprotective agents and no semen resulted in higher proportion (p˂ 0.05) of PMN than the same extenders containing semen (B 43% vs S-B 26.9 % LDL 36% vs S-LDL 25.3%). The duration of significantly elevated PMN was also higher when extenders contained no semen. The extender with no cryoprotective agent (INRA96) also resulted in a higher proportion of PMN (39.6 %) than S-B and S-LDL. DW resulted in a proportion of PMN (44.8%) similar to those of the extenders containing no semen, and higher than those of the extenders containing semen. The results suggest that the presence of semen modulates and reduces the endometrial inflammatory response of jennies. The cryoprotective agents by themselves do not appear to exacerbate the inflammatory response, as a similar response was produced by an extender containing no cryoprotective agent and even by DW.

Evaluation of equine semen frozen in extenders free of animal proteins

Various countries are reticent regarding importation of semen that contains egg-yolk, limiting its commercialization. Egg-yolk composition is complex, varying with the diet of the chicken and egg-yolk granules are similar in size to sperm, interfering with analysis. Therefore, development of a chemically defined cryopreservation extender that maintains seminal parameters is relevant. Twelve ejaculates from 4 stallions of proven fertility (n= 4; r=3) were diluted in one of 5 extenders: 1) EDTA-glucose with 11% lactose, Equex, 20% egg-yolk and 5% dimethylformamide (EY); 2) casein salt and cyclodextrin cholesterol extender (CE); 3) CE with 3% dimethylformamide (CE-3); 4) OptiXcell (OP); 5) BioXcell (BIO). Cryopreservation was carried out in a Cryogen, Neovet automatic freezing machine: -0.5°C/minute to 5°C, 180 minutes equilibration, then -5°C/minute to -20°C and -20°C/minute to -100°C, then plunging in N2. Thawing was carried out at 37°C for 1 minute and sperm kinematic parameters (CASA, AndroVision, Minitube), viability and acrosome status (FITC-PNA-PI stain), membrane lipoperoxidation (BODIPY stain) and DNA fragmentation (SCD) were evaluated. Data were analyzed using ANOVA or Friedman test and results are reported as mean ± SD. Higher (P<0.05) percentages of total and progressively motile sperm were observed in samplescryopreserved in EY (29.25 ± 11 and 21.17 ± 10), CE-3 (30.4 ± 15 and 22 ± 12) and OP (23.1 ± 14 and 15.5 ± 10) compared to those in CE (4.75 ± 3 and 1.58 ± 2) and BIO (8 ± 4 and 3.25 ± 3). Straight-line, curvilinear and average-path velocity parameters, amplitude of lateral head and beat-cross frequency were significantly higher in samples frozen with EY, CE-3 and OP compared to CE and BIO media. Straight-line and average-path velocities were also significantly higher in OP compared to EY medium. Live acrosome-intact sperm were significantly higher in EY (45 ± 14) compared to the rest of the extenders; with CE-3 (25 ± 10) being significantly higher than CE (8 ± 5), OP (9 ± 5) and BIO (13 ± 5). Live, non-peroxidized sperm were significantly higher in EY (47 ± 15) compared to the other extenders; with CE-3 (29 ± 15) being significantly higher than CE (14±10), OP (21 ± 15) and BIO (23 ± 14). No significant differences were observed in the percentages of sperm with intact DNA in any of the extenders: EY (95 ± 4); CE (95 ± 2); CE-3 (93 ± 8); OP (94 ± 7) and BIO (94 ± 5). To conclude, the casein salt and cyclodextrin cholesterol extender with 3% dimethylformamide, without egg-yolk could be a suitable alternative for extenders with animal proteins. Further studies are ongoing to evaluate in vivo fertility of ejaculates cryopreserved in this extender.

Fortification of Extender with Basella rubra Fruit Extract Enhances the Cryosurvival of Ram Semen.

This study aimed to evaluate the impact of Basella rubra fruit extract (BR-FE) on cryopreserved ram sperm's motility, velocity, and membrane integrity. Thirty ejaculates collected from 3 fertile rams (10 from each) were diluted with semen dilution extender (SDE) in a ratio (1:2) and centrifuged to remove 50% supernatant. The remaining sample was mixed with semen cryopreservation extender (SCE) in 1:4 ratio. Then 1.2 mL of SCE diluted sample was divided in four aliquots (0.3 mL each) that were further extended with [(1) control group (0.7 mL of SCE), (2) BR-FE-0.6% group (0.7 mL of SCE supplemented with 0.6% BR-FE), (3) BR-FE-0.8% group (0.7 mL of SCE supplemented with 0.8% BR-FE), and (4) BR-FE-1.6% group (0.7 mL SCE supplemented with 1.6% BR-FE)]. All extended samples were cooled gradually from 25°C to 4°C in half an hour. The 0.1 mL sample from all aliquots was analyzed for precryopreservation sperm parameters and the remaining sample was loaded in 0.5 mL plastic semen straws, cooled gradually to -20°C, and then dipped in liquid nitrogen. After 24 hours of cryopreservation, the straws were thawed for postcryopreservation sperm evaluations. The results (analysis of variance based) showed significantly enhanced percentage of post-thaw sperm membrane integrity, progressive motility, and velocity in BR-FE-0.6% group at both pre- and postcryopreservation stages as compared with all other groups. However, analysis of covariance revealed concentration-dependent cryoprotective effect of BR-FE with maximum percentage of sperm membrane integrity in the 1.6% group. According to these results, BR-FE supplementation adds enormous sperm protective potential to ram sperm cryopreservation medium.