Cryopreservation Of Ram Semen Research Articles

Effect of fumigation height and time on cryopreservation of ram semen

The cooling rate is a crucial factor in the process of freezing semen, influencing the overall freezing effectiveness. The height and time of fumigation can significantly impact the rate of cooling. Appropriate cooling rates can help minimize the formation of ice crystals in spermatozoa and reduce potential damage to them. Therefore, the aim of this study was to evaluate the effect of different fumigation heights and time for the cryopreservation of Hu ram semen. Experiments I–IV assessed the effect of semen cryopreservation by testing the post-thawed spermatozoa total motility (TM), progressive motility (PM) and kinetic parameters fumigated at distances of 2, 4, 6 and 8 cm for durations of 5, 10, 15 and 20 min, respectively. Based on the results of experiments I to IV, experiment V evaluated the effect of semen cryopreservation by testing the post-thawed spermatozoa TM, PM, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level fumigated at distances of 2, 4, 6 and 8 cm for duration of 20 min. The results indicated that fumigation at 2 cm for 20 min significantly (P < 0.05) improved spermatozoa TM, PM, mean angular displacement (MAD), plasma membrane integrity and acrosome integrity compared to other groups. Additionally, it significantly (P < 0.05) reduced spermatozoa ROS level compared to the 6 and 8 cm groups. In conclusion, fumigation for 20 min at a distance of 2 cm from the liquid nitrogen surface is the most suitable cooling method for the cryopreservation of Hu ram semen.

Cryopreservation of ram semen: baicalein efficiency on oxidative stress, chromatin integrity, viability and motility post thaw.

Baicalein (B) has potential antioxidant properties, but it has not been tested as a ram semen extender. This study aimed to assess the impact of B on various sperm parameters and determine its potential influence on semen quality after the freeze-thawing process. During the breeding season, ejaculates were obtained from four rams with the aid of an artificial vagina. The collected mixed semen samples were divided into four groups: control (C; 0), B0.5 (0.5 mM), B1 (1 mM), and B2 (2 mM). After semen extension, the samples were loaded into 0.25 mL straws and stored for 2 h at 4°C prior to freezing in liquid nitrogen vapor and thawed in a water bath at 37°C. Among the groups, B0.5 demonstrated the highest progressive motility results, while B1 and B2 exhibited reduced motility (p < 0.05). In terms of high mitochondrial membrane potential, plasma membrane and acrosome integrity, and viability, B0.5 showed significantly superior outcomes to the other B groups (p < 0.05), although it was not significantly better than C. B1 displayed the highest plasma membrane integrity levels (p < 0.05). Notably, B2 displayed the lowest total antioxidant status levels among the groups (p < 0.05). The findings of this study suggested that the in vitro spermatological characteristics of ram spermatozoa such as progressive motility and chromatin integrity can be protected from the freeze-thawing process by using the 0.5 mM dose of baicalein as a semen extender. The treatment of sperm freezing might benefit from further in-depth research on the role of B in the improvement of cryoinjury and its underlying processes.

The beneficial effects of quinoa seed extract supplementation on ram sperm quality following cryopreservation

Although cryopreservation is a reliable method used in assisted reproduction to preserve genetic materials, it can stimulate the occurrence of oxidative stress, which affects sperm structure and function. This research was conducted to explore the effects of quinoa seed extracts (QSE) on ram sperm quality, oxidative biomarkers, and the gene expression of frozen-thawed ram sperm. Semen samples were diluted in extenders supplemented with 0 (QSE0), 250 (QSE1), 500 (QSE2), 750 (QSE3), and 1000 (QSE4) µg of QSE /mL, and then frozen according to the typical procedure. The findings indicate that the QSE3 and QSE4 groups provided the optimal results in terms of sperm viability and progressive motility. Sperm kinematics were considerably enhanced in the QSE3 group compared to the other groups (P<0.01). QSE (500–1000 µg/mL) significantly decreased the apoptosis-like changes (higher viable and lower apoptotic sperm) in ram sperm (P<0.001). The percentage of live sperm with intact acrosomes was significantly increased, while the percentage of detached and intact acrosomes in live and dead sperm were significantly decreased respectively by the QSE addition (P<0.001). All QSE groups had higher TAC and lower MDA and H2O2 levels than the control group (P<0.001). The expressions of SOD1, CAT, GABPB1, and GPX1 genes in sperm samples were significantly increased, while the CASP3 gene was significantly decreased in all QSE-supplemented samples. Our data suggest that QSE has beneficial effects on sperm quality of cryopreserved ram semen, which are achieved by promoting sperm antioxidant-related genes and reducing apoptosis-related gene.

Effect of Different Dilution Methods and Ratios of Ram Semen on Sperm Parameters after Cryopreservation.

The dilution method and ratio were tested to assess their effects on the Hu ram semen after cryopreservation. Experiment I aimed to explore the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I (Tris-based and egg yolk) under the condition of 1:1 dilution of diluent II (diluent I and glycerol) on the Hu ram semen preserved in liquid nitrogen regarding spermatozoa motility and kinetic parameters. Experiment II aimed to investigate the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I under the condition of 1:2 dilution of diluent II to the Hu ram semen for cryopreservation on spermatozoa motility and kinetic parameters. The purpose of experiment III is to assess the effect of various dilution methods and ratios on the cryopreservation of Hu ram semen by detecting spermatozoa motility, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level. Experiment III includes four groups: one-step dilution method and two-step dilution method. The two-step dilution method includes two groups: 1:2, 1:1 and 1:3, 1:2, and the one-step dilution method includes two groups: 1:5 and 1:11. The results indicated that the post-thawed spermatozoa total motility (TM), progressive motility (PM) and average motion degree (MAD) were highest in the 1:2 group and significantly higher (p < 0.05) than those in the 1:1 and 1:4 groups under the condition of 1:1 dilution of diluent II. The post-thawed spermatozoa TM and PM of the 1:3 group were significantly higher (p < 0.05) than those of the other groups under the condition of 1:2 dilution of diluent II. The post-thawed spermatozoa TM, PM, plasma membrane integrity and acrosome integrity of the two-step group (1:3, 1:2) were the highest and significantly higher (p < 0.05) than those in the other groups. Additionally, the post-thawed spermatozoa ROS level of the two-step group (1:3, 1:2) was significantly lower (p < 0.05) than that in the one-step groups (1:5 and 1:11). Therefore, a two-step dilution (1:3, 1:2) was found to be the most suitable method and ratio for diluting the Hu ram semen after cryopreservation.

Effects of organic and inorganic selenium supplementation on frozen-thawed ram semen at two cooling periods.

The aim of this study was to evaluate the effects of different selenium compounds on the sperm quality of cryopreserved ram semen. Ejaculates from four rams, collected using an artificial vagina heated to 38°C, were individually evaluated. The approved ejaculates were pooled and diluted (1:1 v:v) in Tris-egg yolk extender (20%, v/v) and separated into two control groups, one cooled for 2h and the other for 4h. The pooled ejaculates at the two cooling periods were supplemented with two doses (0.5 and 1μg/mL) of organic selenium (ORG), and inorganic selenium (SeNa), each. The samples were packed in 0.25ml straws, at a concentration of 400 × 106 sperms/mL and stored in liquid nitrogen. The straws were thawed in a water bath at 37°C for 20s, and the samples were subjected to sperm kinetics evaluation by Computer Assisted Semen Analysis software. Sperm membrane integrity, acrosome morphology, and mitochondrial potential were assessed. In addition, oxidative stress markers reactive oxygen species (ROS), ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive species (TBARS), and glutathione peroxidase (GPx) enzyme activity) were also evaluated. No significant improvement was observed in the ram semen quality at the two cooling times. Supplementation of the freezing extender with 0.5μg/mL ORG, subjected to 4h cooling period, increased the sperm motility when compared with the control group at the same cooling time. In addition, the 0.5μg/mL SeNa group, under the 2h cooling period, showed an increase in sperm motility when compared to the control group at the same cooling period. Considering the importance of sperm motility as a fertility parameter, our study indicates that supplementation with ORG and SeNa can help improve the total motility of the cryopreserved ram semen.

The effect of different concentrations of laminarin on the quality of cryopreserved ram semen

BACKGROUND: Increasingly, sheep breeders are using artificial insemination to produce lambs, so finding methods that preserve ram sperm can be useful. OBJECTIVE: To determine the protective effects of different concentrations of laminarin on ram sperm motility, viability, abnormalities, membrane, and DNA integrity, superoxide dismutase enzyme (SOD) activity, and malondialdehyde (MDA) production after freeze-thawing. MATERIALS AND METHODS: The ejaculates of four rams were collected and stored at 35 °C. Semen samples were diluted with a tris-base extender containing 100, 200, 400, and 800 μg/mL of laminarin and a control extender containing no laminarin, then frozen in liquid nitrogen after 4 h in the refrigerator. RESULTS: In the treatment of frozen-thawed spermatozoa with 800 μg/mL laminarin, motility, viability, membrane integrity, and DNA integrity were significantly higher than in the control. In spermatozoa that were exposed to 800 μg/mL laminarin after thawing, MDA production was significantly lower than in the control group. The percentage of abnormal spermatozoa in 800 μg/mL laminarin was significantly lower than that in the control. CONCLUSION: The addition of 800 μg/mL laminarin to the freezing extender increases motility, viability, SOD activity, and plasma membrane integrity, while reducing abnormality and MDA production in freeze-thawed ram semen.

Comparative characteristics of protocols for cryopreservation of ram semen

Purpose: study and comparison of protocols for cryopreservation of sperm of stud rams.Every day the popularity of sheep farming in Russia is increasing, since sheep are unpretentious in maintenance and at the same time they are multiproduct animals, providing the population with the necessary raw materials (wool, felt) and food products (meat, milk). For this reason, artificial insemination is of fundamental importance in the development of this branch of livestock farming, an important part of which is the correct cryopreservation of ram sperm. There are quite a few advantages of using cryopreserved sperm. Cryopreservation of sheep sperm contributes to the spread of the use of artificial insemination in this branch of the livestock industry. Freezing technology largely ensures the preservation of genetic material and its transportation, eliminating geographical barriers in the use of artificial insemination, and allows increasing the load on the ram sire without risk to his reproductive health. However, during the process of cryopreservation, sheep sperm receives irreversible damage due to cold shock, osmotic stress and oxidative processes, which lead to a decrease in the ability to fertilize sperm. Thus, chromatin may be damaged, membrane permeability increased, hyperoxidation and the formation of reactive oxygen species are possible, which affects the ability to fertilize. As a result, frozen ram semen may have low fertility. For this reason, it is extremely important to select an up-to-date freezing and thawing protocol with maximum recovery of viable and functional sperm of a given animal species for successful further use in artificial insemination. Technological advances in various fields of biotechnology have led to improvements in protocols, methods and equipment used in the laboratory, which have a positive impact on the reliability, accuracy of the procedure. This article summarizes research on the effects of different cryopreservation procedures, cooling rates, cooling times and thawing protocols on ram semen quality. In addition, a correct protocol for cryopreservation of Romanov breed sperm was formed, based on the results of our own research.

Correlation between fatty acids levels in chicken, duck, goose, pigeon, quail and turkey egg yolks and post-thawed quality of ram semen.

The comparison between adding egg yolks (EY) of chicken, duck, goose, pigeon, Japanese quail or turkey to the Tris glycerol extender on the quality of ram semen before freezing and post-thawing was evaluated. The correlation between fatty acids levels in egg yolks of different avian species and the post-thawed quality of ram semen was studied. The pooled ejaculates collected from five rams were extended with Tris EY glycerol extenders containing the EY of chicken, duck, goose, pigeon, quail or turkey and cryopreserved at -196°C. The straws were evaluated before freezing and post-thawing for sperm motility using a sperm cell analyser, vitality using a FluoVit kit and abnormality using a SpermBlue stain besides plasma-membrane and DNA integrities using a hypo-osmotic swelling test and a Halomax kit, respectively. The moisture, ash, protein and fatty acid (FA) contents of EY of chicken, duck, goose, pigeon, quail and turkey were analysed using a gas chromatograph. The chicken and quail EY extenders significantly improved the total progressive motility (32.05 ± 1.41 and 31.68 ± 1.43, respectively), vitality, plasma membrane and DNA integrities and abnormalities of post-thawing ram semen in comparison with other EY extenders. Pigeon EY had the lowest saturated fatty acids (SFAs) in comparison with the other types of EYs. The chicken and turkey EYs had the lowest percentage of (monounsaturated fatty acids) MUFAs in comparison with the other types of EYs. The highest percentage of polyunsaturated fatty acids (PUFAs) was observed in the turkey, pigeon and chicken EYs which were considered double or triple their percentage in duck and goose EYs, respectively. Significant positive correlations existed between SFAs levels and total motility, vitality, plasma membrane functionality and DNA integrity (0.77, 0.80, 0.67, 0.52, respectively). Significant negative correlations existed between gondoic EY levels and total motility, vitality, plasma membrane functionality and DNA integrity. In conclusion, the EYs of duck, goose, pigeon or turkey cannot substitute the chicken EY in ram semen extenders as they gave lower post-thawing quality. The quail EY can be used as a good replacer for chicken EY in the extender used for cryopreservation of ram semen. The EY composition of FAs can significantly affect the quality of ram semen post-thawing.

The mitochondria-targeted antioxidant “MitoQ” preserves quality and reproductive performance of ram spermatozoa cryopreserved in soybean lecithin-based extender

Cryopreservation of ram semen is helpful for distributing proved spermatozoa for reproductive goals, but cold shock has destructive effects on fertility ability of frozen sperm cells. This study was performed to investigate the effect of the novel mitochondria-targeted antioxidant “MitoQ” on ram sperm quality and fertility potential during cryopreservation process. Semen samples were diluted in extenders supplemented with 0, 1, 10, 100 and 1000 nM MitoQ and then frozen according to the standard protocol. Motility and velocity characteristics, lipid peroxidation, acrosome integrity, membrane functionality, mitochondria active potential, viability, apoptosis status, DNA fragmentation, ROS concentration and reproductive performance were evaluated after thawing. In results, 10 and 100 nM MitoQ presented higher (P ≤ 0.05) total motility, progressive motility, average path velocity, acrosome integrity, membrane functionality, mitochondria active potential and viability as well as lower (P ≤ 0.05) lipid peroxidation, apoptosis status, DNA fragmentation and ROS concentration compared to the control group and the other treatments. Moreover, after fertility trial, 10 and 100 nM MitoQ resulted in higher (P ≤ 0.05) pregnancy, parturition and lambing rates than control group. Therefore, MitoQ is able to preserve quality parameters and fertility potential of post-thawed spermatozoa in sheep and it could be an effective additive for supplementation of ram's semen cryopreservation medium during reproductive programs.

Methionine, cysteine, and butylated hydroxytoluene enhance cryosurvival of ram semen on post-thaw and post-incubation time points.

Despite there have been many experiments conducted about antioxidants, the best sole or combination use of antioxidants to include as a standard ingredient to freezing extenders is yet to be found. This study was designed to investigate the different doses of methionine (2.5 and 5mM), cysteine (1 and 2mM), and butylated hydroxytoluene (BHT) (1 and 2mM) for ram semen cryopreservation on post-thaw and post-incubation (6h) time points over spermatological parameters. Semen samples were collected from Kivircik rams via electro-ejaculator in breeding season. After essential spermatological evaluations, appropriate samples were pooled then split into 7 equal aliquots to create study groups (antioxidant free control, 2.5mM methionine, 5mM methionine, 1mM cysteine, 2mM cysteine, 1mM BHT, and 2mM BHT). Semen samples were put into French straws (0.25mL), and freezing procedure (two-step) was conducted via a programmable gamete freezer. At both time points, motility, HOST, PSA-FITC, and TUNEL assays were made to discover the impacts of cryopreservation and incubation process over sperm cells. Antioxidant supplemented groups yielded better results compared to the control groups in terms of various spermatological parameters not only at post-thaw time point but after incubation for 6h of time. The study demonstrated that supplementing sperm freezing extenders with previous antioxidants may create new approaches to cryopreservation procedures, and through increasing success rate of freezing, fertility results may increase to better results in near future.

Association between different soy lecithin-based extenders and freezing rates in ram semen cryopreservation

The aim of the study was to evaluate the effect of the association between glycine-milk (GM) based extenders made with different concentrations of soy lecithin (SL) and freezing rates (FR) on semen quality after thawing. Pooled semen from rams (n = 12) were diluted in GM extenders with 20% egg yolk (EY–20%) or with different concentrations of SL: 0.5% (SL–0.5%), 1.0% (SL–1.0%), and 2.0% (SL–2.0%). The diluted semen (150 ×106 spermatozoa/0.25 mL) was frozen at three FR of − 10, − 20, and − 60 °C/min, and subsequently thawed and analyzed. Results revealed that EY–20% and SL–2.0% had better kinetic parameters, and showed higher proportions of viable, non-apoptotic, plasma-membrane-intact spermatozoa (A-/PI-) and non-capacitated spermatozoa (F), and had lower acrosome-reacted spermatozoa (AR) in the EY–20% and satisfactory values for SL-2.0% compared to SL–0.5% and SL–1.0% (P < 0.05). The FR at − 20 and − 60 °C/min maintained higher A-/PI- and viable spermatozoa compared to − 10 °C/min. The combination EY–20% and − 60 °C/min showed the highest A-/PI- and F (P < 0.05) and the lowest AR, and it did not differ from the combinations EY–20% at − 20 °C/min and SL–2.0% at − 20 °C/min (P > 0.05). In conclusion, the combination EY–20% and − 60 °C/min, showed the best cryoprotective effects on ram spermatozoa. Changes in spermatozoa after thawing were related to the use of the type of extender, the amounts of the same compound in the extender, and the freezing rates to which they were subjected.

Proteomic analysis reveals the potential positive effects of Mito-TEMPO on ram sperm motility and fertility during cryopreservation

The aim of this study was to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on the protein profile of ram sperm during cryopreservation and evaluate the cryoprotective roles of Mito-TEMPO on ram sperm quality and fertilization capacity. Semen collected from 8 Dorper rams was cryopreserved in TCG-egg yolk extender supplemented with various concentrations of Mito-TEMPO (0, 20, 40 and 60 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The cervical artificial insemination (AI) was performed to evaluate the fertilization ability of cryopreserved ram sperm. The alterations of sperm proteomic profile between the control and MT40 groups were determined using iTRAQ-coupled LC-MS. Supplementation with 40 μM of Mito-TEMPO resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed ram sperm were elevated in the MT40 group. The inclusion of 40 μM Mito-TEMPO in freezing extender also resulted in the higher pregnancy rate of ewes. A total of 457 proteins including 179 upregulated proteins and 278 downregulated proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05. Sixty-one DEPs with (FC > 1.5) were dramatically regulated by Mito-TEMPO. These DEPs are mainly involved in sperm motility, energy metabolism and capacitation. Our data suggest that the beneficial effects of Mito-TEMPO on sperm motility and fertility potential of cryopreserved ram semen are achieved by regulating sperm antioxidant capacity and sperm proteins related to energy metabolism and fertility.

COMPARISON BETWEEN RAPID AND SLOW CRYOPRESERVATION PROTOCOLS FOR RAM SEMEN

Information regarding adequate freezing protocols of ram semen considering freezing distance and time during cryopreservation has not been adequately reported. Therefore, this study aimed to compare two freezing protocols for Najdi ram's semen. In the rapid freezing protocol, the straws were frozen at 5 cm over the surface of liquid nitrogen for 15 minutes. While in the slow freezing protocol, the straws were frozen at 8 cm over the surface of liquid nitrogen for 20 minutes. The semen was collected from five rams and extended with tris egg yolk glycerol cryodiluent. The extended semen was chilled slowly to 50C within two hours and equilibrated for two hours before being frozen on the liquid nitrogen vapor and cryopreserved at -1960C. There was no significant (P &gt;0.005) effect of the freezing protocol on the sperm’s total motility, plasma membrane integrity, DNA integrity, and abnormalities. However, the vitality, fast progressive motility, straight-line velocity, average pathway velocity, linearity, and wobble were significantly higher in rapid freezing than in slow freezing protocol. In conclusion, cryopreservation of ram semen using rapid freezing (5 cm for 15 mins) protocol was better than slow freezing (8 cm for 20 mins) protocol regarding post-thawing semen quality. Key words: freezing protocol; cryopreservation; egg yolk; ram; semen

Ram Semen Cryopreservation for Portuguese Native Breeds: Season and Breed Effects on Semen Quality Variation

The semen quality is one of the determinant factors of ram semen cryopreservation. The present retrospective study aimed to characterize the seasonal ram pattern during the year for ten Portuguese local sheep breeds, hypothesizing that the breed and season had low effects on the main spermatozoa traits. A total of 1471 ejaculates were used and evaluated (fresh semen) from 85 rams between 2004 and 2020 and re-evaluated after thawing (thawed semen). The effect of breed, season, and sperm cryopreservation on nine semen traits were evaluated. The volume per ejaculate, spermatozoa (SPZ) concentration, and total number of SPZ per ejaculate, were affected by breed (p < 0.001) but not by season (p > 0.05). As expected, the semen processing was the most significant (p < 0.001) factor of variation on seminal parameters. Moreover, breed and interactions between breed × semen processing, modulated the response of alive SPZ, abnormal morphology, head, and intermediate piece defects. In fresh semen, season only affected the intermediate piece defects due to the highest percentage observed between February and April period in some breeds. Overall, and despite the mentioned particularities, there were similarities among the ten local breeds. We also concluded that the seasonal effect on ejaculate and SPZ traits is not significant in our region. These local ram breeds have low seasonality and can be employed in natural mating as well as semen donors for cryopreservation and assisted reproductive biotechnologies during the whole year at our latitude.

Addition of orange, pineapple and beet juices as extenders for cryopreservation of ram semen

Resumo Em busca de melhorias na criopreservação do sêmen, substâncias naturais são comumente estudadas com o objetivo de melhorar a qualidade do sêmen. O objetivo deste estudo foi avaliar o efeito da adição de sucos de laranja, abacaxi e beterraba em diferentes concentrações e combinações ao diluidor de criopreservação de sêmen ovino. Foram utilizados cinco ejaculados de cinco carneiros adultos. O pool de sêmen foi diluído em diluente à base de gema de ovo e misturado com os seguintes 15 tratamentos (na concentração final de 400x10⁶ sptz/ml): laranja 10% (O10) e 15% (O15); abacaxi 10% (P10) e 15% (P15); beterraba 10% (B10) e 15% (B15); abacaxi + laranja 10% (PO10) e 15% (PO15); abacaxi + beterraba 10% (PB10) e 15% (PB15); beterraba + laranja 10% (BO10) e 15% (BO15); abacaxi + beterraba + laranja 10% (PBO10) e 15% (PBO15); e o grupo controle (CON). Pós-descongelação em palhetas de 0,25 ml a análise da qualidade do sêmen criopreservado foi realizada pelo CASA e citometria de fluxo. A análise de variância foi realizada e as médias comparadas pelo teste SNK. O teste de correlação de Pearson também foi realizado. Nenhum efeito foi observado na adição de sucos ao diluidor de sêmen antes da criopreservação. Após o descongelamento, embora estatisticamente semelhante ao grupo controle, a motilidade total do grupo B10 atingiu padrões aceitáveis de motilidade total. Além disso, o grupo B10 apresentou os maiores valores (p&lt;0,05) de motilidade progressiva que o grupo controle ou os outros tratamentos. A adição de 10% de suco de beterraba ao diluente de sêmen ovino pode melhorar a criopreservação da motilidade espermática.

Adição de sucos de laranja, abacaxi e beterraba em diluidor para criopreservação de sêmen de carneiros

Abstract Searching for improvements in semen cryopreservation, natural substances are commonly studied focusing to improve the sperm quality. The aim of this study were evaluated the effect of adding orange, pineapple, and beet juices in different concentrations and combinations to the ram semen cryopreservation extender. Five ejaculates from five adult rams were used. The semen pool was diluted in egg yolk-based extender and mixed with the following 15 treatments (at a final concentration of 400.106 sptz/mL): orange 10% (O10) and 15% (O15); pineapple 10% (P10) and 15% (P15); beet 10% (B10) and 15% (B15); pineapple + orange 10% (PO10) and 15% (PO15); pineapple + beet 10% (PB10) and 15% (PB15); beet + orange 10% (BO10) and 15% (BO15); pineapple + beet + orange 10% (PBO10) and 15% (PBO15); and the control group (CON). Post-thaw in 0.25 mL straws semen quality analysis of cryopreserved semen was performed by CASA and flow cytometry. Analysis of variance (PROC GLM) was carried out and the averages were compared using the SNK test. Pearson's correlation test was also performed. No effect was noted in the addition of juices to the semen extender prior to cryopreservation. Post-thawed, although, statistically similar to the control group, the total motility of the B10 group reached acceptable standards of total motility. In addition, B10 group showed the highest values (p&lt;0.05) of progressive motility than control group or other treatments. The addition of 10% beet juice to the ram semen extender can improve the cryopreservation of sperm motility.

Does Rainbow Trout Seminal Plasma-Cysteine Combination Affect the Cryo-Survivability and Post-Thaw Incubation Resilience of Ram Semen?

The aim of this study was to evaluate the effect of both pure rainbow trout seminal plasma (RTSP) supplementation and RTSP-cysteine combination on cryopreservation success and post-thaw incubation resilience of ram semen in the nonbreeding season. For this purpose, different doses of RTSP (0%, 1%, 10%, and 15%) with or without cysteine supplementation were used for experiments. Ejaculates chosen for experiments were pooled and then divided into eight equal volumes for grouping (Control-ControlC, RTSP1-RTSP1C, RTSP10-RTSP10C, and RTSP15-RTSP15C). After cryopreservation, frozen-thawed semen samples were incubated for 5 hours at 37°C for determination of post-thaw incubation resistance. Motility, HOST, TUNEL, Rh123-PI, and CTC tests were performed at 0 hour and 3rd and 5th hours of post-thaw incubation to evaluate the efficacy of all experimental groups. The RTSP10 and RTSP10C groups were noted to provide the best protection on motility, plasma membrane integrity, DNA integrity, and mitochondrial function of cryopreserved ram semen. On the other hand, the best protection against cryo-capacitation was observed in RTSP15 and RTSP15C groups. The addition of cysteine was found to be effective when the higher (15%) or lower (1%) doses of RTSP were used, as well as for no use of RTSP.

Ram semen cryopreservation: Effects of egg yolks from different avian species

Ram semen cryopreservation: Effects of egg yolks from different avian species

Can Honey Improve the Quality of Cryopreserved Cross Bred Ram Semen Added to Tris Egg Yolk Extender?

BACKGROUND: Honey can improve the quality of cryopreserved ram semen because of its multinutrient and cryoprotective nature added to standard tris egg yolk extender. OBJECTIVE: Different concentrations of honey were added to the standard tris egg yolk extender to improve the post-thaw quality of crossbred ram semen. METHOD: Thirty six (36) ejaculates from eight healthy cross bred rams were pooled and divided into four aliquots. Standard tris egg yolk extender without any alteration acted as Control (C) and was supplemented with different concentrations of honey, viz. T1 (honey 1.5%), T2 (2.5%), and T3 (3.5%). RESULTS: The percent (mean ± S. E. M) sperm motility at pre-freeze was significantly (P&lt;;0.05) higher in Group T2 and at post-thaw in Group T3 in comparison to T1 and C treatment groups. The percent (mean ± S. E. M) HOST reacted spermatozoa at post-thaw was significantly (P&lt; 0.05) higher in Group C and at pre-freeze the value was significantly (P&lt; 0.05) higher in the same treatment group than Group T1. The mean MDA level (mean ± S. E. M) at post thaw was significantly (P&lt;0.05) low er in Group T3 than the treatment groups C and Group T1. CONCLUSION: From this study it is concluded that the addition of 3.5% honey to the standard tris egg yolk extender provides better protection to ram semen than the addition of 1.5% honey (i. e., Control).

The Supplementation of Bee Bread Methanolic Extract to Egg Yolk or Soybean Lecithin Extenders Can Improve the Quality of Cryopreserved Ram Semen.

Bee bread has numerous nutritional benefits and bioactive compounds. Other bee byproducts have been used as extender additives to improve semen cryopreservation. Here, we examined the effects of supplementing egg yolk extender (EYE) or soybean lecithin extender (SBLE) with bee bread extract (BBE) on the quality of cryopreserved ram semen. Semen was collected from five adult Rahmani rams once a week for 7 weeks. EYE and SBLE were supplemented with BBE. Antioxidant capacity and total phenolic compound, total flavonoid compound, and total soluble carbohydrate levels of BBE were measured. Sperm characteristics, including progressive motility, viability, abnormalities, membrane integrity, and acrosome integrity, were analyzed after equilibration, thawing, and thawing followed by a 2-h incubation. The total antioxidant capacity and malondialdehyde, hydrogen peroxide, aspartate transaminase, alanine transaminase, alkaline phosphatase, and total acid phosphatase levels in extenders were determined after thawing. Sperm apoptosis was analyzed using annexin V assays. SBLE was more effective than EYE for cryopreserving ram semen. Extender supplementation with BBE improved ram semen quality during freezing in a concentration-dependent pattern. Motility, vitality, and membrane integrity were particularly enhanced in BBE-treated semen. Additionally, BBE promoted antioxidant and enzymatic activities and reduced apoptosis in semen. Thus, extender supplementation with BBE improved sperm cryopreservation.