Yolk Extender Research Articles

Effect of turmeric extract and dimethyl sulfoxide on buffalo semen freezability and fertility

The freeze-thaw process results in structural and functional damages caused by overaccumulation of reactive oxygen species (ROS). Addition of antioxidants to semen extenders is of a great importance to overcome this oxidative damage. The study objective was to evaluate the consequence of using Tris-citric acid fructose egg yolk (TCFY) extender supplemented with a combination of turmeric extract and dimethyl sulfoxide (DMSO) [TTD] on sperm freezability and fertility. From five tubes (each containing 5 mL TCFY), the first tube contained neither turmeric extract nor DMSO and was kept as control. The other four tubes contained 1.5% DMSO plus 100 µL/5 mL, 200 µL/5 mL, 300 µL/5 mL and 400 µL/5 mL turmeric extract. Semen samples were pooled and extended to reach an application of 60×106 sperm/mL (TT1D to TT4D, respectively). Diluted semen was exposed to the freezing protocol. The post cooling results revealed significant improvement in percent of alive spermatozoa in TT1D, significant decrease in sperm abnormalities in all concentrations used, significant improvement in intact acrosome percentage in TT1D, TT2D and TT4D. The post thawing results exhibited significant improvement in sperm motility in TT1D, TT2D and TT4D; significant amelioration of sperm membrane integrity (HOST) in TT1D, TT2D, TT3D and TT4D. Acrosome integrity was maintained in all concentrations as in the control. It was concluded that TT1D revealed the best semen quality in cooled semen; and TT1D, TT2D exhibited the superior post thawing semen quality. In addition, conception rate (CR) of the post-thawed semen was ameliorated in TT1D, TT2D, TT3D and TT4D with TT1D being the best one with this regard.

Combination of nanoparticle green tea extract in tris-egg yolk extender and 39 °c thawing temperatures improve the sperm quality of post-thawed Kacang goat semen.

Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x106 sptz/mL) and cryopreserved. Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.

The effects of cysteine and L-carnitine on the DNA integrity of post-thaw sperm of frozen buck semen.

The addition of antioxidants to semen extenders is fundamental to limit oxidative changes resulting from cryopreservation. This experiment was designed to clarify the effects of cysteine and L-carnitine (LC) on post-thaw sperm criteria. Semen samples were collected once weekly from five mature Zaraibi bucks for 10 successive weeks. Fresh semen samples were evaluated for basic semen characteristics, and the accepted samples were pooled and divided into seven aliquots: control (Tris egg yolk extender), cysteine (2.5, 5 and 10 mM) and LC (2.5, 5 and 7.5 mM). Aliquots were diluted and subjected to cryopreservation procedures. After thawing, sperm criteria were evaluated regarding motility, viability, plasma membrane (hypo-osmotic swelling test, HOST) and acrosome integrity, total antioxidant capacity (TAC), level of malondialdehyde (MDA), and DNA comet assay. Addition of both 10 mM cysteine and 7.5 mM LC significantly increased post-thaw sperm motility, viability, HOST, and acrosome integrity. Comet assay revealed significant decreases in DNA damage with best results at 2.5 mM cysteine and 7.5 mM LC. Besides, 10 mM cysteine and 7.5 mM LC exhibited significant diminish in MDA levels. Cysteine was positively correlated with sperm viability, acrosome integrity, and comet, but negatively correlated with MDA and tail length. Positive correlations were declared between LC and progressive motility, viability, HOST and TAC, while negative correlations were found with MDA, comet, tail DNA, tail moment, and olive tail moment. Supplementing cysteine and LC to frozen buck semen improved sperm criteria and decreased DNA damage, possibly due to their effectiveness in the suppression of MDA formation.

The enriched Y-bearing sperm combined with delayed fixed-time artificial insemination for obtaining male Simmental crossbred offspring.

Background and Aim:The production of male calf beef cattle is an agricultural innovation needed to increase the farm’s productivity as a provider of meat sources. This study aimed to determine the sex ratio of the offspring of cows inseminated with Y-bearing sperm enriched by Percoll density gradient centrifugation and swim-up, combined with delayed fixed-time artificial insemination (FTAI).Materials and Methods:Ejaculates of Simmental bulls were divided into four equal portions and grouped as T0 (control, non-sexed semen), T1 and T2 were sexed semen using Percoll density gradient centrifugation three and five levels, respectively, and T3 was sexed semen using swim-up. After the sex was sorted, the semen was diluted in a tris-egg yolk extender, packaged in French mini-straws containing 50 million live sperm cells, and frozen. Pre-sexed, post-sexed, and post-thawed spermatozoa were evaluated based on progressive motility, viability, intact plasma membrane, and abnormality. The post-thawed semen of T0 was artificially inseminated to recipient cows at 12 h after onset of estrus (not delayed FTAI). Meanwhile, the delayed FTAI was conducted 18-20 h after onset of estrus using the T0, the best of T1 and T2, and the T3 post-thawed semen.Results:The Percoll density gradient centrifugation reduced motility, viability, and intact plasma membrane but increased sperm abnormalities. Meanwhile, the swim-up process increased motility, viability, and intact plasma membrane of sperm cells but decreased sperm abnormalities. Post-thawed semen decreased motility, viability, and intact plasma membrane of sperm cells but increased sperm abnormalities. The sex ratio of the Simmental crossbred offspring was 96.08% and 100% in T1 and T3, respectively, compared to 48.25% and 67.39% in T0 not delayed and delayed FTAI, respectively.Conclusion:The Percoll density gradient centrifugation and swim-up methods are prospective for obtaining male offspring.

Effect of Milk Type Subjected to Different Heat Treatments on Cryo-Survivability and In Vivo Fertility of Buffalo (Bubalus bubalis) Spermatozoa in a Milk-Based Extender.

Oxidative stress is a major contributory factor to cellular damage during semen cryopreservation and results in a decreased fertilizing capacity of cryopreserved bull sperm. The inclusion of exogenous antioxidants sometimes exerts deleterious effects on sperm quality. Thus, enhancing the endogenous production of antioxidants is a requirement. This study aimed to investigate the effect of milk type heated at different temperatures on the antioxidant potential of extenders, and the subsequent post-thaw quality parameters and in vivo fertility of buffalo bull semen. Cow (C) and buffalo whole milk (B) were used separately for semen extender preparation, heated at five different temperatures (T1 = 90°C, T2 = 100°C, T3 = 110°C, T4 = 120°C, T5 = 130°C) for 10 minutes. Reactive sulfhydryl groups were measured in each subgroup by Ellman's reagents as CT1 = 143.2 μM, CT2 = 147.4 μM, CT3 = 151.5 μM, CT4 = 157.2 μM, CT5 = 161.8 μM, BT1 = 168.3 μM, BT2 = 172.5 μM, BT3 = 176.7 μM, BT4 = 196.3 μM, and BT5 = 205.7 μM. All semen samples were cryopreserved in milk-based extenders by using standard procedures. Post-thaw quality parameters including total and progressive motility, mitochondrial membrane potential, plasma membrane integrity, and acrosome integrity were found to be higher (p < 0.05) in the group (BT3) containing buffalo milk heated at 110°C, whereas in the same group, lipid peroxidation was found to be lower (p < 0.05) as compared with other treatment groups and control group. In vivo fertility of cryopreserved buffalo sperm was compared among BT3, CT1 (conventionally used milk extender), and a Tris egg yolk extender group. The fertility rates [47% (54/114), 30% (33/108), and 36% (37/103)] were higher (p < 0.05) in BT3 as compared with other groups. This study suggests that buffalo milk heated at 110°C has high antioxidant potential and improves post-thaw quality and in vivo fertility of cryopreserved buffalo bull semen.

Freezing ability of two slow permeating cryoprotectants on rooster semen diluted with tris egg-yolk juice extender

Cryo-damage reduction with slow permeating cryoprotectants has been identified as a promising approach for poultry semen preservation. This study was conducted to evaluate the efficacy of dimethylformamide (DMF), methylacetamide (MA), and their mixtures (DMF+MA) in the cryopreservation of rooster semen extended with tris quail egg-yolk (TEY) extender supplemented with orange or tomato juice. The extender was prepared by adding 25% of egg-yolk in tris buffer. 18% &amp; 13% (v/v) DMF, MA, and (DMF+MA) were added to the extender constituting six treatments. Semen was collected from fifteen roosters of 35weeks of age by backstroke massage of mid abdomen followed by semen pooling. Pooled semen was divided into six fractions and two steps dilution was done. Extended semen samples were subjected to cryopreservation procedures and finally stored at -196 oC. After 48h, semen samples were thawed at 5oC and evaluated for motility, viability, membrane integrity, and acrosome integrity. Effects of juices on TEY extended semen were further evaluated with 13% and 18% (DMF+MA). 15% of orange (O) and tomato (T) juices were supplemented to TEY extender. Same dilution, freezing and evaluation procedures were observed. Finally, fertility trials were conducted on the best treatments. The results revealed that 13% and 18% (DMF+MA) had significantly (P&lt;0.05) higher motility, viability, and membrane integrity. TEYO-18% (DMF+MA), TEYO 13%(DMF+MA+O), and TEYT 13% (DMF+MA) had significantly (P&lt;0.05) higher motility and membrane integrity compared to others. 100% fertility rate was also recorded for 13%(DMF+MA). It was concluded that 13% (DMF+MA) with or without juice supplementation is beneficial

A 150 kDa Protein Derived from Bull Seminal Plasma Extended the Survival Time of Kacang Goat Sperm Stored at 5°C.

Artificial insemination has proven to be an effective method for increasing population size and genetic quality of Kacang goats. However, innovation is required to maintain the quality of Kacang goat semen in storage. This study aimed to examine the effects of supplementing the 150 kDa protein assumed as IGF-I complex derived from bull seminal plasma in skim milk-egg yolk extender on the quality of Kacang goat sperm stored at 5°C. Twelve ejaculates collected from three Kacang goats were divided into three groups. In the control group (T0), the ejaculates were extended with skim milk-egg yolk only. In the treatment groups (T1 and T2), the ejaculates were extended with skim milk-egg yolk supplemented with the IGF-I complex protein at 12 μg and 24 μg/100 mL, respectively. The extended semen was stored at 5°C, and the viability, motility, intactness of the plasma membrane, malondialdehyde concentration, and apoptotic sperm percentage were evaluated daily for five days. The results showed that the T1 was the most effective treatment for maintaining Kacang goat semen at a quality acceptable for artificial insemination over five days of storage at 5°C. However, the T0 and T2 groups retained acceptable qualities for only three days at 5°C. It could be concluded that supplementation of 12 μg of the 150 kDa protein derived from bull seminal plasma per 100 mL extender successfully extended the life span of Kacang goat sperm for five days.

Maintaining the Quality of Kacang Buck Semen in Chilled Storage with the Addition of Green Tea Extract in Extender

Kacang goats can be bred through artificial insemination techniques using liquid semen from a superior buck. The study was aimed to determine the quality of Kacang buck semen in chilled storage when green tea extract (GTE) was added to the semen extender. Specifically, 12 ejaculates from three Kacang bucks were diluted in skim milk–egg yolk extender containing 0 mg, 0.05, 0.10, and 0.15 mg of GTE/100 mL. The extended semen was stored at 5°C, and its quality was evaluated daily for 5 d. Comparatively, semen with 0.10 mg of GTE/100 mL had the best quality (p<0.05): it had the highest sperm viability, sperm progressive motility, and sperm intact plasma membrane values, and the lowest malondialdehyde levels and DNA fragmentation percentage. However, as the storage period increased, there was a decrease in sperm viability, sperm motility, and sperm intact plasma membrane values with an increase in malondialdehyde levels and DNA fragmentation (p<0.05). The sperm motility of semen with 0.10 mg of GTE/100 mL was maintained during 5 d of chilled storage to meet the qualification for artificial insemination. In contrast, the sperm motility of semen with 0.05 mg and 0.15 mg of GTE/100 mL was maintained for 4 d, whereas that of the control semen was maintained only for 3 d. Thus, the addition 0.10 mg of GTE/100 mL of skim milk-egg yolk extender seems to help maintain the quality of Kacang goat sperm in chilled storage.

Preservability and conception of chilled and cryopreserved semen with Triladyl egg yolk extender in dromedary camels (Camelus dromedarius)

Artificial insemination (AI) technology has not shown much advancements in camels, attributable to various difficulties of semen preservation and insemination in the species. The present study intended to explore the preservability of camel semen under chilled and frozen states, improve proficiency of AI steps and assess conception to AI. Over the two years of study, 383 ejaculates collected from five adult male camels were preserved in Triladyl egg yolk extender using refrigeration or liquid nitrogen and used for AI trials in camels subjected to induction of ovulation. Under chilled storage, even though few samples retained satisfactory motility even beyond 96 h, a majority of the samples suffered significant drop in motility within 24 h. Out of 239 and 739 freezing trials (FT) respectively in the first and second seasons, 18.83% and 23.00% FT achieved a post-thaw motility (PTM) of at least 40%. Insemination studies showed progressive decrease in conception rate with the duration of chilled storage since fresh extended and 24 h chilled semen samples produced 32% and 3.7% conception respectively. Cryopreserved semen achieved two pregnancies (1.82%) out of 110 inseminations in the second season. It is concluded that camel spermatozoa suffers rapid depletion of conception chance upon preservation and underlying reasons needs to be investigated. However, the achievement of two pregnancies indicates the possibility of conception and the scope for cryopreservation based AI technology in dromedary camels.

The The Efectiveness of Jamblang (Syzygium cumini) Leaves Extract Inclusion Skim Milk-Egg Yolk Extender on Motility and Viability of Aceh Cattle Spermatozoa during Pre-Freezing and Post-Thawing

The process of semen freezing causes an increase in free radicals concentration which can damage spermatozoa. The addition of natural ingredients in semen diluent is expected to solve this challenges. One of the natural ingredients that can be used is jamblang (Syzygium cumini) leaves. The objective of the current study was to investigate the quality of spermatozoa in Aceh cattle which was added with jamblang leaves extract in skim milk- egg yolk extender during pre-freezing and post-thawing. This study applied Completely Randomized Design (CRD) with 5 treatments and 5 replications. The treatments consisted of J0 = skim milk-egg yolk; J1 = skim milk-egg yolk + jamblang leaves extract 0.2%; J2 = skim milk-egg yolk + jamblang leaves extract 0.4%; J3 = skim milk-egg yolk + jamblang leaves extract 0.6%; and J4 = skim milk-egg yolk + jamblang leaves extract 0.8%. The parameters observed in this study were the percentage of motility and viability of frozen semen of Aceh cattle. The data obtained were analyzed using Analysis of Variance (ANOVA) and if differences were found, then it would be continued with Duncan's Multiple Distance test. The results showed that the addition of jamblang leaves extract in egg yolk skim milk significantly affected the percentage of motility during pre-freezing and post-thawing, significantly affected spermatozoa viability during pre-freezing and significantly affected the spermatozoa viability during post-thawing. J3 treatment (jamblang leaves extract 0.6 gram/100 ml) it should be higher than the other treatment, where the percentage of motility at pre-freezing and post-thawing were 55.48% and 52.71%, respectively, and the percentage of viability during pre-freezing and post-thawing were 56.59% and 53.94%, respectively. It was concluded that the addition of jamblang leaves extract in the skim milk-egg yolk extender affected the percentage of spermatozoa motility and viability of Aceh cattle during pre-freezing and post-thawing.

Effect of Hydrated Carbon 60 Fullerene on Frozen Ram Semen Quality.

The aim of this study was to evaluate the effect of hydrated carbon 60 fullerene (C60HyFn) on ram semen quality during cryopreservation. Three ejaculates from each of seven Akkaraman rams were collected using an artificial vagina during the nonbreeding season and pooled. Pooled semen samples were divided into 10 equal parts and diluted with tris + egg yolk extender not containing (control) and containing 100, 200, 400, and 800 nM and 1, 5, 10, 20, and 40 μM C60HyFn at 37°C. After addition of 5% glycerol and an equilibration process for 3 hours, the samples were frozen in 0.25-mL straws in an automatic freezing device at -140°C and stored in a liquid nitrogen container. Straws were thawed 24 hours after freezing and analyzed immediately with no incubation period. Motility, kinematic parameters, abnormality, vitality, hypo-osmotic swelling test (HOST), and oxidative stress levels were analyzed in thawed semen. Compared with the control, 200, 400, and 800 nM and 1 and 5 μM C60HyFn doses increased motility and HOST values and decreased the dead sperm rate. When compared with the control, addition of C60HyFn significantly decreased malondialdehyde levels (between 200 nM and 40 μM doses) and significantly increased glutathione peroxidase (between 800 nM and 40 μM doses) and catalase (between 1 and 40 μM doses) activities. In conclusion, results of this study show that the C60HyFn nanoparticles are nontoxic to ram semen and their supplementation in the extender is beneficial to sperm motility and membrane integrity after freeze-thawing.

Zinc oxide nanoparticles alter the membrane potential of mitochondria from post-thawed ram spermatozoa

• Zinc oxide nanoparticles increases mitochondrial membrane potential of spermatozoa from ram semen. • Sperm kinetics are not altered by zinc oxide nanoparticles. • Zinc oxide nanoparticles do not protect the plasma membrane of ram sperm during freezing. The objective of this study was to evaluate how the addition of zinc oxide nanoparticles (nano-ZnO) to freezing extender affect the characteristics of post-thawed ram semen. For this research, seminal pools from three Santa Inês rams were used. Each seminal pool was diluted in Tris-egg yolk (5 % glycerol) extender, supplemented with nano-ZnO (0, 10, 50, 100 or 200 μg/mL), at the final concentration of 200 × 10 6 spermatozoa/mL. Subsequently, samples were filled in 0.25-mL straws, frozen in an automated system and stored in liquid nitrogen (−196 °C). At the time of analyses, semen samples were thawed in a water bath at 37 °C for 30 s and processed for evaluation. All samples were evaluated for sperm kinetics by computer-assisted sperm analysis (CASA), as well as assessed for mitochondrial membrane potential (MMP), plasma membrane integrity (PMi) and acrosomal membrane integrity (ACi), by epifluorescence microcopy. The sperm kinetics results showed that when adding nano-ZnO (10, 50, 100 and 200 μg/mL) to freezing extender, there were no differences (P > 0.05) in post-thawed ram semen between control and treatment groups, nor among the treated groups themselves. It is emphasised that all groups treated with nano-ZnO presented more gametes (P ≤ 0.05) with high MMP than the control group. However, no differences were observed (P > 0.05) in the percentage of sperm with PMi between control and treatment groups (nano-ZnO). In conclusion, the addition of nano-ZnO (10, 50, 100 or 200 μg/mL) to freezing extender increases MMP but does not alter the kinetics or protect membranes of ram spermatozoa after the freezing-thawing process.

The Protective Impacts of α-tocopherol Supplementation on the Semen Quality of Sapera Goat Preserved at 4oC

Supplementation of vitamin E (α-tocopherol) as an antioxidant into an extender is useful to maintain sperm quality during the process of cryopreservation by protecting the sperms from reactive oxygen species (ROS). This study aims to evaluate the protective impacts of vitamin E supplementation on the motility, viability, and reducing the abnormality of sperms in Sapera goats. The sample of semen used in the present study was obtained from fresh semen and good-quality ejaculates of the Sapera goats collected by an artificial vagina. The groups involve one controlled treatment group, which contains semen and tris egg yolk (TEY) extender without vitamin E (P0), and three treatment groups that contain semen, TEY extender, and different doses of vitamin E, i.e., 1 mg/mL (P1), 2 mg/mL (P2), and 3 mg/mL (P3), respectively. Data obtained from the experiments were analyzed using Repeated Measures Multivariate Analysis (MANOVA). The results showed that TEY extenders added with vitamin E at a dose of 1 mg/mL of vitamin E (P1) produced the highest motility and viability with the lowest abnormalities of sperm after 24th hour of cryopreservation. The lowest sperm motility and viability among the treatment groups was observed in the TEY extender added with vitamin E at a dose of 3 mg/mL (P3) at 96th hour after cryopreservation, which was almost the same as the control group without vitamin E (P0) that had the lowest motility and viability. In conclusion, the current study found that supplementation of vitamin E as an antioxidant at a dose of 1 mg/mL in TEY extender can preserve semen of the Sapera goats in refrigerator up to 96 h at 4°C and provides higher sperm motility and viability percentage, with a lower percentage of sperm abnormality.

Effect of insulin-like growth factor-1 complex of Simmental bull seminal plasma on post-thawed Kacang buck semen fertility.

Background and Aim:Kacang buck sperm is cryosensitive due to the seminal plasma of semen itself. Meanwhile, bull seminal plasma contains the insulin-like growth factor-1 (IGF-1) complex, which is cryoprotective. The addition of the crude protein of Simmental bull seminal plasma increased the quality of post-thawed semen of Kacang buck. The study was conducted to determine the effects of Simmental bull seminal plasma with IGF-1 on the fertility of post-thawed Kacang buck semen.Materials and Methods:Buck semen was diluted in the following skim milk-egg yolk extender preparations: Without the addition of Simmental bull seminal plasma IGF-1 complex protein (T0); with the addition of 12-μg Simmental bull seminal plasma IGF-1 complex protein (T1); and with the addition of 24-μg Simmental bull seminal plasma IGF-1 complex protein (T2). The extended semen was packed in 0.25-mL straws and frozen. Post-thawed semen fertility was evaluated based on the following variables: Sperm motility, viability, intact plasma membrane (IPM), malondialdehyde (MDA) levels, capacitation status, and acrosome reaction. The difference in each variable among the groups was evaluated using analysis of variance, followed by Tukey’s honestly significant difference test, at a 95% level of significance. Meanwhile, principal component analysis (PCA) was used to identify the principal component of semen fertility among the seven parameters.Results:The T1 group showed the highest sperm motility, viability, IPM, and percentage of incapacitated sperm and the lowest MDA levels, percentage of capacitated sperm, and acrosome reaction. PCA revealed that sperm motility had a moderate to very robust correlation with other variables and is the most crucial parameter, accounting for 80.79% of all variables.Conclusion:The IGF-1 complex in Simmental bull seminal plasma was useful for increasing the fertility of post-thawed Kacang buck semen, and sperm motility was the principal component of semen fertility.

Effects of different ultrastructures of lecithin on cryosurvival of goat spermatozoa.

This study was to evaluate the effects of two different ultrastructures of lecithin including nanoparticles (NPE mostly nanomicelles) and lecithin nanoliposome (NLE) with egg yolk extender (EYE) on goat sperm cryopreservation. Semen samples were collected from 6 goats, then pooled, diluted and then frozen. Motility and motion parameters, plasma membrane integrity and functionality, morphology, apoptosis status (Annexin V-PI), acrosome integrity, DNA fragmentation and in vitro fertilisation were assessed. Total motility and most motion parameters were higher in EYE (p<.05) compared with the two lecithin extenders, while there were no significant differences between NLE and NPE. NLE and NPE had higher values for viable spermatozoa (Annexin V-PI) (p<.05) compared with EYE. The highest value for dead spermatozoa was observed in EYE (p=.08). A higher percentage of DNA fragmentation (p<.05) was detected in EYE compared with NPE. Plasma membrane integrity and functionality, morphology, acrosome integrity and fertility of spermatozoa indicated no significant differences between extenders. Data suggested that ultrastructural changes of lecithin (micelles versus. liposome) could not improve the sperm cryosurvival of goat spermatozoa. Moreover, we cannot also claim that lecithin-based diluent supplies better protection compared with the egg yolk in goat.

Carboxymethyl cellulose and glycerol act synergistically as cryoprotectant during cryopreservation of ram semen

Wider implementation of AI in sheep in the field condition has not been possible till date due to very poor conception rate after cervical insemination with cryopreserved semen. Poor cervical penetrability in ewe and diminished sperm functions in cryopreserved semen are considered responsible for it. In the present study, effect of carboxymethyl cellulose (CMC) on post-thaw qualities of ram semen was investigated. Ejaculates from eight adult Malpura rams were pooled and diluted (800 × 106 sperm mL−1) with TES-Tris-fructose-egg yolk extender having either 5 or 6% glycerol and supplemented with 0, 0.25, 0.5, 0.75 and 1.0% (w/v) CMC and packaged into 0.25 mL French mini straws. The straws were progressively cooled to 5 °C inside a cold cabinet (5 °C) and then equilibrated for 22 h inside a refrigerator (2–5 °C). Straws were frozen at −25 °C min−1 up to −125 °C using a programmable cell freezer (Planer Biomed R-204, UK) and finally plunged into liquid nitrogen. The post-thaw progressive motility was higher (P < 0.05) in 0.75% CMC-treated group compared to control. Overall, both pre-freeze and post-thaw sperm kinetics was comparable between CMC-treated and control groups. The post-thaw sperm viability, acrosomal integrity and sperm with high mitochondrial membrane potential (hMMP) were relatively higher while sperm with high membrane cholesterol was significantly (P < 0.05) higher in presence of 0.25% CMC compared to the control. Both sperm having hMMP and non-capacitated sperm were significantly (P < 0.05) higher in presence of 5% glycerol than 6% glycerol. Similarly, functional membrane integrity (FMI) was higher in presence of 5% glycerol than 6% glycerol when CMC was added at 0.5% to extender. In conclusion, both 0.25% CMC and 5% glycerol resulted in improvement in several post-thaw sperm functions in cryopreserved ram semen. Thus CMC demonstrated cryoprotective effect on ram sperm in a synergistic manner with glycerol.

Is addition or removal of seminal plasma able to compensate for the dilution effect of buffalo semen?

The present study aimed to compensate dilution effect using additional seminal plasma (SP) in conventional (80 million (M) spermatozoa/ml) dose and low spermatozoa/dose (8M spermatozoa/ml). We also attempted to confirm whether removal of SP before the extension of ejaculates affects post-thaw sperm quality of buffalo semen. For this, semen ejaculates (N=15) were divided into four groups: control (CON), removal of SP by centrifugation (NSP), resuspension of the centrifuged semen pellet into SP (CEN) and extra supplementation of SP (ESP). All groups were diluted into two different semen doses to 20 and 2M spermatozoa/0.25ml using tris egg yolk extender and subsequently cryopreserved. We found that neither addition nor removal of SP affected sperm motility, kinematics, longevity, mitochondrial superoxide production and high mitochondrial membrane potential (MMP). Further, the addition or removal of SP was not able to compensate dilution effect in 2M groups resulting in a significantly (p<.05) reduction in sperm motility, kinematics, sperm longevity, membrane integrity, MMP, and an increase production of mitochondrial superoxide. In conclusion, it appears that role of SP in the sperm cryopreservation process is insignificant.

Kinematics motility of friesian-holstein sperm sexing in l-ascorbic acid treatments

The study aimed to examine the kinematics motility and recovery rates of frozen-thawed Friesian Holstein bull sexing semen in the addition of l-ascorbic acid treatment. Semen collected from Friesian Holstein bulls with the minimum standard of sperm motilities for sexing sperm (60%), and abnormality (<20%) used for this study. Semen was diluted with final concentration 300x106 sperm/ml in Brackett-Oliphant (BO) medium. The sperms were sorted using the BSA column method. Sexing sperm were extended with Tris egg yolk (TEY) extender in addition L-Ascorbic Acid (LA) 0%, 0.25%, 0.5%, and 0.75% before freezing process. Frozen sexing semen was thawed for the kinematics motility evaluation with parameter; Tmot, Pmot, VAP, VCL, VSL, LIN, ALH, BCF, DAP, DCL and DAP using CASA system also recovery-rate evaluation. The results showed kinematics motility evaluation for 0.75% TEY-LA had lowest value (P<0.05) in Tmot, Pmot, velocity and distance compared to control group, 0.25% and 0.5% TEY-LA. The recovery rate 0.5% TEY-LA had highest values between among all treatment in X and Y frozen sexing semen. In conclusion, the addition of 0.5% l-ascorbic acid to tris egg yolk extender could be advantageous tool to improve the kinematics motility and increased recovery rates of Friesian Holstein sexing sperm.

Effect of trolox added to freezing extenders over goat and ram spermatozoa

Semen cryopreservation is responsible for decrease the gamete fertility, due to structural and functional damages. Among the various causes, oxidative stress, resulting from the higher generation of reactive oxygen species (ROS), has been attributed to affect semen quality. Thus, it was objectified evaluate the effect of Trolox on ram and goat sperm, subjected to freezing. Semen pools of goat (n=5) and ram (n=6) were diluted in skimmed milk (7% glycerol) or Tris-egg yolk (5% glycerol) extender, respectively, added or not of Trolox (0, 20 or 40 μM/ml) and frozen. After thawing (37 °C/30 s), aliquots of semen were evaluated for lipid peroxidation by high performance liquid chromatography, coupled with a photodiode array detector (HPLC-DAD), and flow cytometry (C11-BODIPY581/591), besides of plasma membrane and acrosome integrity by fluorescence microscopy, and sperm kinetics by computerized sperm analysis (CASA). The antioxidant treatment with Trolox did not determine significant effects (p&gt;0.05) on lipid peroxidation, plasma membrane integrity, acrosomal integrity and on the kinetic parameters evaluated. Thus, it is concluded that Trolox (20 or 40 μM) did not have a protective or deleterious effect on goats and ram sperm, submitted to freezing.

Substitution of Egg Yolk with Arabic Gum in Extenders Used for Ram Semen Cold Storage or Cryopreservation

The current research was designed to compare the effects of substitution of egg yolk (EY) in ram extender with Arabic gum (AG) in different concentration in cooled or frozen semen collected by artificial vagina (AV) or electro-ejaculator (EE) in Noemi rams. Semen was collected 2 times per week for 6 weeks from 6 rams by EE method and after a rest period of 2 weeks, semen samples were collected by AV method. Tris extender either contained 15% EY (Control) or 3%, 5% or 7% AG (AG3, AG5 and AG7, respectively) were used as semen extenders. Spermatozoa were evaluated after cold storage (4 °C) or freezing/thawing in liquid nitrogen for sperm motility parameters: total motile spermatozoa (% TMS), rapid progressively motile sperm (% PRS), curvilinear velocity (VCL) μm/s, rectilinear velocity (VSL) μm/s, the average path velocity (VAP) μm/s, straightness index (% STR), linearity coefficient (% LIN), morphological defects and acrosome membrane integrity. Results showed that AV-collected sperm and diluted with AG or EY were more efficient in motility than EE. The values of VCL, VSL and VAP were higher in AG3 and AG5 and EY extenders for semen collected by AV compared to EE. After cold storage, AG3 gave better (P<0.01) results of LIN and STR either in spermatozoa collected by AV or EE. Significant differences in morphology defects were noted in semen collected by EE regardless of extenders used, however, AG supplementation enhance morphological defects in spermatozoa collected by AV (P<0.05). Post-freezing, Tris extender containing EY and AG5 in AV-collected spermatozoa had higher TMS, PRS, VCL, VSL and VAP compared to others. Sperm morphological defects were higher in AG7 extender in EE and EY extender in AV group. Plasma membrane integrity was significantly (P<0.05) higher in AG3 and AG5 extenders in AV-group compared to control and other groups. The acrosome membrane integrity in AV-semen groups was greater (P<0.05) than that of EE-semen group. It can be concluded that, Arabic gum is a good candidate to replace egg yolk in ram semen extender when added at a concentration of 3 or 5%. Although method of artificial vagina semen collection gave better results than electro-ejaculator. However, Arabic gum could be added to ram semen extender to enhance the electro-ejaculator-collected semen characteristics when added at a concentration of 5%