L-carnitine added to post-thawed semen acts as an antioxidant and a stimulator of equine sperm metabolism.

The objective of this study was to enhance the in vitro sperm quality and in vivo fertility of frozen-thawed equine semen by the addition of l-carnitine (LC) to post-thawed semen. Different concentrations of LC were added to thawed samples to obtain four treatments control and 0.5, 1 and 2 mM LC. In the in vitro experiments, sperm motility and kinematics, membrane integrity and intracellular calcium ion concentration ([Ca2+ ]i ) were investigated, and the antioxidant bioactivity of LC was assessed by measuring hydrogen peroxide and nitrite concentrations (NO2 - ). The fertility rate was assessed via the artificial insemination of mares. The treatment with 1 mM LC increased sperm [Ca2+ ]i (60.6 ± 0.05 AU), reduced nitrite concentration (39.1 ± 14.9 µM/µg protein), increased the sperm straightness percentage (STR: 78.3 ± 5.3%) and increased the pregnancy rate (75%) as compared to the control ([Ca2+ ]i 48.4 ± 0.05 AU, NO2 - concentration 63.1 ± 14.4 µM/µg protein, STR 67.5 ± 7.9%, 12.5% pregnancy rate, p < 0.05). These results suggest that 1 mM LC acts as an antioxidant and stimulator of sperm metabolism in post-thawed equine semen, increasing the fertility rate. Thus, addition of LC might be an alternative to improve the fertility of poor quality post-thawed equine semen.

Association of linear type pelvic traits with testicular characteristics and reproductive capability of breeding dairy bulls.

Associations of pelvic linear type traits (PLT) on production and reproduction of cows were widely documented; in contrast, importance of PLT on bulls' reproductive ability, semen quality, semen cryo-preservability, frozen semen doses (FSD) production etc. was inadequately reported. The present study was conducted on Frieswal bulls (N = 378, age: 6-91 months, m) to assess age-wise growth dynamics of pelvic dimensions, classifying age into 6 groups (6 m, 7-12 m, 13-24 m, 25-36 m, 37-48 m, >48 m). Iso-age group bulls' (N = 100; 25-36 m) records were analysed after classifying seasons (summer/rainy/winter) of semen collection, if pelvic morphometric traits had any practical associations with testicular traits, semen quality and FSD production or not. A discriminant function was developed based on PLT, which showed poor predictive ability to distinguish FSD/non-FSD category bulls. Age significantly influenced growth and dimensions of gonadal and external pelvic morphometry traits. The dimensions of PLT increased at higher rate up to 36 m age and thereafter enhancement became insignificant. Pelvic linear type traits were positively (p < .01) associated with testicular/scrotal traits. Among PLT studied, pelvic triangle area (PTA) was the strongest discriminating variable to distinguish between good and poor breeding bulls. Bulls of larger PTA (≥1000 cm2 ), at average 30 m age, produced relatively inferior-quality semen, viz. lower volume (-3%), sperm concentration (-48 × 106 /mL), motility, semen quality index, total sperm counts/ejaculate (-601 × 106 ), motile sperm counts/ejaculate (-474 × 106 ) and total post-thaw motile sperm counts (-226 106 ), than bulls of lesser PTA (<1000 cm2 ). It was concluded that external PTA could be a useful addition to the bulls' breeding soundness evaluation.

Non-gonadal linear type traits can discriminate reproductive ability of breeding dairy bulls.

Linear type traits are easily measurable phenotypic characteristics that help breed characterization, selection of animals for breeding and found to be associated with animals' performance. Unlike cows, there have been limited studies linking body linear traits with male reproductive ability and semen cryo-preservability of breeding bulls. Present study reported the age-related changes in body linear type traits in Frieswal (N = 378) dairy bulls and its relevance with reproductive potentials of breeding bulls. Our results indicated that body frame size traits were significantly and positively correlated with gonadal linear traits. Among the selected body mophometric parameters body length, chest girth and head circumference were the important body linear type traits having capability to discriminate between bulls of frozen semen doses (FSD) and non-FSD categories. Discriminant function has been developed based on body linear traits of crossbred dairy bulls to find out males of superior reproductive potentials. Our finding provided evidence that body length (humerous tuberosity to tuber ischii) was the most powerful linear body trait associated with breeding bulls' reproductive ability and semen quality.

Effect of organic selenium dietary supplementation on quality and fertility of cryopreserved chicken sperm.

Oxidative stress due to cryopreservation has been considered as a major factor in sperm damage. Supplementation of the diet with different concentrations of organic selenium has been proposed to improve the quality of fresh and frozen-thawed semen in different breeds of roosters. Sixteen Pradu Hang Dum (Thai native) and 16 Rhode Island Red roosters were used in this study. Four levels of selenium supplementation between 0 and 0.9 ppm were examined. After 14 days of feeding, semen samples were collected twice a week and the fresh semen was evaluated. Then semen from each group was pooled and cryopreserved. The fertility of frozen-thawed semen was determined by inseminating 48 layer hens. Supplementation of diets with 0.3, 0.6 and 0.9 ppm selenium improved the fresh semen in terms of sperm viability and normal morphology (P < 0.01). Sperm concentration increased (quadratically, P < 0.001) with increasing dietary selenium levels. Meanwhile, post-thawed semen quality in terms of sperm motility, viability, live with intact acrosome and functioning mitochondria improved significantly with selenium treatments of 0.6 and 0.9 ppm, and lipid peroxidation was decreased (P < 0.001) and fertility improved (P < 0.05) with those levels of selenium treatment. In addition, there were differences between breeds with respect to some fresh or frozen semen quality parameters (P < 0.05). In conclusion, the breed affected both fresh and frozen semen. Even there were no statistically significant differences in the parameters from groups 0.6 and 0.9 ppm on frozen-thawed semen quality, but the highest sperm concentration was found in 0.6 ppm. Therefore selenium supplementation of diets at 0.6 ppm was recommended to improve the quantity and quality of fresh and frozen semen.

Intravaginal insemination depth influences fertility outcomes in Indian red jungle fowl (Gallus gallus murghi).

The depth of intravaginal insemination to achieve optimum fertility with frozen-thawed semen is highly species specific in birds and differ even in breed and/or strains of a species. Therefore, study was designed to evaluate the influence of intravaginal insemination depths (2 and 4 cm) on fertility outcome in Indian red jungle fowl. Semen collected from eight mature cocks was pooled, diluted in extender and cooled to 4 °C. Glycerol (20%) was added to chilled semen, equilibrated for 10 min and cryopreserved. After 3 days of storage, frozen semen was thawed in water bath at 37 °C for 30 s. After glycerol removal, intravaginal Inseminations were performed at the depth of 2 and 4 cm. The no. of fertilized eggs (31.4 ± 1.6 vs. 27.7 ± 1.8), fertility rate (65.7 ± 3.6 vs. 58.8 ± 4.0), no. of hatched chicks (27.8 ± 1.9 vs. 23.5 ± 1.6), hatchability of set eggs (58.8 ± 4.3 vs. 49.7 ± 3.2) and hatchability of fertilized eggs (88.4 ± 2.8 vs. 84.3 ± 2.2) were recorded higher with intravaginal depth of 4 cm compared to 2 cm. It is concluded that intravaginal insemination at the depth of 4 cm enhances the fertility outcomes of the frozen-thawed Indian red jungle fowl semen.

Use of nutraceuticals in the stallion: Effects on semen quality and preservation.

Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from different sources, vitamins and antioxidants, carnitine and botanical extracts. Different nutraceuticals have been highlighted to improve stallion fertility by providing optimal levels of antioxidants, with the most promising results obtained by the combination of PUFAs and antioxidants that resulted to be essential for the maintenance of normal reproductive functions and the reduction of cryodamage in cooled and frozen equine semen.

Genetic and non-genetic factors affecting conception rate of frozen semen in small holder dairy farmer system of rural India.

Fertility traits are as important as production traits in crossbred bovine population. Assessment of the fertility of bull in any frozen semen production program and evaluation of the conception rate under actual field conditions provide valuable information. The objective of this retrospective study was to estimate the effect of genetic and non-genetic influence on conception rate of frozen semen bulls maintained at Bharatiya Agro-Industries Foundation (BAIF) Pune based on the conception rate in cattle population of small holder dairy farmer system. The data comprising of 1,08,238 insemination records pertaining to 83 Holstein Friesian pure and crossbred bulls available at BAIF Pune were used to analyze conception rate. The fixed effect solutions and covariance components were estimated by linear mixed model using the restricted maximum likelihood method in WOMBAT software. The genetic correlations were estimated using bivariate analysis between post thaw motility and conception rate. The study was based on fertility related information from cows maintained in different villages of India and thus reflects the actual fertility of frozen semen used. The study was suggestive of influence of very small fraction of genetic effect and higher impact of management effect on conception rate. Fertility-related information available from this study is an invaluable asset in decision making process of breeding policies.

Comparison of TNC and standard extender on post-thaw quality and in vivo fertility of Thai native chicken sperm.

Semen extender has a vital role in preservation of sperm cells properties in terms of sperm viability, motility, acrosome integrity, and mitochondrial membrane potential. The objective of the present study was to evaluate a new extender, known as Thai native chicken (TNC) extender compared to BHSV-based and modified Sasaki extenders for freezing chicken semen. Semen from Thai native roosters was collected, pooled, and randomly divided into three groups. Semen was frozen with a simple freezing method using nitrogen vapor and dimethylformamide. In the first experiment, post-thaw motion parameters, viability, acrosome integrity, mitochondrial function, and lipid peroxidation levels were analyzed using computer-assisted sperm analysis, propidium iodide, fluorescein isothiocyanate-conjugate peanut agglutinin, JC-1, and the thiobarbituric acid reaction. Results showed that the type of extender had no effect on the percentage of total motile and curvilinear velocity. The percentage of progressive motile, straight-line velocity, and average path velocity of post-thawed semen were significantly lower in TNC compared to the modified Sasaki extender. However, the percentages of post-thawed acrosome integrity and active mitochondria were significantly higher in TNC extender (P < 0.05). For the second experiment, semen was thawed by using each of extenders thereafter, was inseminated to 48-layer breeder hens to determine the fertility rate. Among the three extenders used, the highest fertility rate was found in TNC extender. In conclusion, TNC extender can be recommended as an appropriate and useful cryopreservation media for native chicken semen since it maintains the quality of rooster semen and fertility after freezing and thawing process.

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca).

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ±â€¯7.7%) and membrane functionality (60.5 ±â€¯4.2%) and higher mitochondrial activity (21.5 ±â€¯3.7%) than those preserved in ACP-117c (20.9 ±â€¯5.4% motile sperm; 47.1 ±â€¯2.5% functional membrane; 11.8 ±â€¯1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ±â€¯3.3%) in comparison to samples diluted in ACP-117c (18.6 ±â€¯1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.

An investigation of the time period within which frozen-thawed semen delivers a high conception rate in lactating dairy cows.

We determined the length of time within which frozen-thawed semen delivered a high conception rate in present-day lactating dairy cows. The cows utilized were a total 100 milking Holstein-Friesian cows kept in tie-stall style farms. We carried out artificial insemination (AI) during the periovulatory period at a predicted time based on ovulation, and checked ovulation at 6-h intervals after AI. The period from AI to ovulation ranged from 48 h (i.e., 48 h before ovulation) to -12 h (i.e., 12 h after ovulation). High conception rates averaging 63.0% were obtained by carrying out AI > 6-30 h before ovulation, significantly higher than the conception rates of 30.0% (P < 0.05) and 26.9% (P < 0.01) from AI carried out earlier than 30 h before ovulation and later than 6 h before ovulation, respectively. It was concluded that frozen-thawed semen delivers a conception rate of ≥ 60% for > 24-30 h after AI, and that a conception rate of ≥ 60% can be achieved by carrying out AI 6-30 h before ovulation using frozen-thawed semen.

Birth of first foals through embryo transfer after artificial insemination using frozen semen in Japan.

Until now, there have been no reports of foals born through embryo transfer after artificial insemination using frozen semen in Japan. The aims of this study were to develop a riding crossbred horse and evaluate the prospects of embryo transfer technology in multiplying horse population. In both donor and recipient mares, luteolysis was induced by the administration of 0.1 mg Cloprostenol to synchronize the onset of estrus, and ovulation was induced by administering 2000 IU human chorionic gonadotropin (hCG) or 0.75 mg Deslorelin. Frozen semen from an Irish Connemara pony stallion was used to breed a Hokkaido native pony mare by deep-horn artificial insemination (dose, 400 × 106 sperm). A non-surgical technique was used to collect embryos from the donor mare at day 7 post-ovulation and transfer them transcervically into the uterus of recipient mares (n = 4) immediately after collection. Weekly blood samples were collected from the recipients throughout pregnancy. A total of four embryos were recovered from seven collection attempts (57% recovery) from a donor mare in a single breeding season. Three of the four transferred embryos maintained successful pregnancy and delivered a healthy live foal (75% birth). A normal progesterone profile was observed throughout gestation in recipient mares. In conclusion, for the first time, to the best of our knowledge, this study describes the birth of foals through non-surgical transcervical embryo transfer in Japan after artificial insemination using frozen semen. We expect that this new crossbreed (Connemara pony × Hokkaido native pony) will be a good riding breed.

Practical experience with artificial insemination (AI) using fresh chilled and frozen semen in mares.

The objective of this study was to compare the efficiency of artificial insemination (AI) carried out with frozen and fresh, diluted and chilled semen under field conditions. One hundred and twenty-nine mares of different breeds were included in the study. Eighty-one out of the 107 mares inseminated with fresh, chilled semen got pregnant. Seven pregnant mares aborted and 74 foals were born. Out of the 22 mares inseminated with frozen semen, 17 mares got pregnant. Two mares out of the 17 pregnant mares aborted and finally 15 healthy foals were born. No difference was found between the two groups in the ratio of the foals born (P > 0.05). The comparison of medians for the number of insemination cycles did not show significant differences. However, a significant difference (Kruskal-Wallis test, P = 0.014) was found in the number of the inseminations per conception in favour of frozen semen (2.5 vs. 1.8 with fresh chilled and frozen semen, respectively). The Cox regression revealed that the type of semen has a significant impact (P < 0.001) on the service period (duration of the insemination period): the use of frozen semen prolonged the insemination period. This could be due to management issues, since re-insemination with frozen semen took place after only one/a few missed oestrous cycles not used for AI.

Effects of season and single layer centrifugation on bull sperm quality in Thailand.

OBJECTIVE: The aim of study was to investigate the effects of season and single layer centrifugation (SLC) before cryopreservation on post-thaw bull sperm quality in Thailand. METHODS: Semen was collected from 6 bulls (Bos indicus) in summer, rainy season and winter 2014 through 2016. Semen characteristics, sperm morphology, sperm kinematics, viability, chromatin structure and mitochondrial membrane were evaluated. Meteorological data were available from the local meteorological station. RESULTS: Season had an effect on semen characteristics in the raw ejaculate, with higher proportions of normal spermatozoa and lower abnormalities in winter than in the other two seasons. Sperm kinematics, viability, DNA fragmentation index, and mitochondrial membrane potential were not different between seasons. Sperm samples selected by SLC had greater normal morphology and a lower proportion with bent tails than controls and higher values of progressive motility (PRO), beat cross frequency, linearity, straightness, wobble (WOB), and lower values of slow motility, velocity average path (VAP), velocity curved line, and amplitude of lateral head displacement than controls. In addition, SLCselection had a favorable effect on PRO, VAP, and WOB that differed among seasons. CONCLUSION: Our results suggested that these bulls were well adapted to their location, with season having an effect on sperm morphology. Moreover, SLC could be used prior to cryopreservation, regardless of season, to enhance normal morphology and kinematics of bull sperm samples without adversely affecting other parameters of sperm quality. However, there was considerable variation among bulls in DNA fragmentation index, mitochondrial membrane potential and sperm viability. In addition, SLC had a positive effect on sperm morphology and sperm kinematics, which could be expected to influence fertility.

Presence of Round Cells Proteins do not Interfere with Identification of Human Sperm Proteins from Frozen Semen Samples by LC-MS/MS.

In sperm proteomic experiments round cells and leukocyte proteins are profiled along with sperm proteome. The influence of round cell and leukocyte proteins on the sperm proteome has not been investigated. The objective of this study was to identify if the proteins from round cells, including leukocytes, interfere with the proteomic analysis of spermatozoa in frozen semen samples. Proteomic profiling of sperm was performed using liquid chromatography-tandem mass spectrometry in four groups: Group 1 contained neat semen with round cells and leukocytes ≥ 1 × 106/mL, group 2 contained neat semen with round cells ≥ 1 × 106/mL that was processed by 65% density gradient to remove the round cells and leukocytes, group 3 contained neat semen with round cells < 1 × 106/mL, and group 4 contained neat semen with round cells < 1 × 106/mL that was processed by 65% density gradient to remove the round cells. Pure leukocyte culture was used as control group. A total of 1638, 1393, 1755, and 1404 proteins were identified in groups 1, 2, 3, and 4, respectively. Comparative analysis of group 1 vs. 3 revealed 26 (1.18%) differentially expressed proteins (DEPs). On the other hand, only 6 (0.31%) DEPs were observed with group 2 vs. 4. Expression of these DEPs were either absent or very low in the control group. The results of our proteomics analysis failed to show any influence of non-spermatogenic round cell proteins on sperm proteome identification. These results validate the use of neat semen samples for sperm proteomic studies.

Effect of breed and other animal-related factors on conception rate to artificial insemination with frozen semen in mares in Ethiopia.

Equine reproduction is unique by having long behavioral estrus and differences in time of breeding between breeds and individuals of mares. An experimental study was conducted at the Balderas Sport Horses and Recreational Center, Addis Ababa, Ethiopia, from January to June, 2018, to evaluate conception rate to frozen semen in local and exotic crossbreed mares. Mares were teased to characterize estrus behavior and examined by ultrasound in determining imminent ovulation. Inseminations were done post ovulation within an average of 6-9 h using frozen-thawed semen. The overall conception rate to frozen semen was 15/21 (71.43%) with 8/11 (72.73%) in crossbreed and 7/10 (70%) in local breed mares. Age and body condition score (BCS) of animals had no significant effect on conception rate to AI with frozen semen. A slightly higher conception rate was obtained when ovulation was from the right ovary than when ovulated from the left ovary. A higher conception rate was obtained when the diameter of the preovulatory follicle was ≤ 45 mm than above diameter. The conception rate increased significantly with increased number of services/conception with an overall mean ± (SEM) of 2.2 ± 0.2 services/conception. A more number of services/conception were required for local breed (2.7 ± 0.2) than crossbreed mares (1.8 ± 0.3) and again for lower body condition scores than higher condition scores of mares. In conclusion, the increased number of services improved the conception rate with significant difference between breed of mares, whereas good management of mares for improved body conditions could be required to decrease the number of services per conception.

Dimethylacetamide and trehalose for ram semen cryopreservation.

Eighteen semen samples from nine Santa Ines rams were cryopreserved in order to study the cryoprotective capacity of dimethylacetamide (DMA) at two concentrations (3% and 6%), and its interaction with trehalose (TRE, 100 mOsmol). A further objective of this study was to evaluate in vivo fertility of ram semen cryopreserved with dimethylacetamide. The control treatment was an extender medium with 6% glycerol (GLY). Five treatments were examined: 6% GLY, 3% DMA, 6% DMA, 3% DMA + TRE, and 6% DMA + TRE. After thawing, the kinetic sperm parameters were analyzed using a computerized system. Sperm viability was observed using a multiple parameter sperm staining with propidium iodide (for plasmatic membrane integrity), JC-1 (for mitochondrial membrane potential), and FITC-PSA (for acrosomal integrity). The isosmotic extender with GLY was the most effective medium for the maintenance of sperm characteristics, compared to extenders containing DMA as cryoprotectant. Regarding fertility, the extender medium with 3% DMA may be a safe alternative for sperm cryopreservation, and does not compromise the fertility rates. The addition of TRE decreased the kinetic sperm parameters; however, a positive effect on the integrity of the plasmatic and acrosomal membranes was observed.

Effect of adding heterologous versus homologous bovine seminal plasma prior to cryopreservation on bull sperm quality after thawing.

SummaryThe aim of this study was to investigate the effect of adding homologous or heterologous bovine seminal plasma (SP) to SP-free sperm samples before freezing on sperm quality after thawing. Ejaculates from bulls of known fertility were used as a source of SP. The SP was removed from further aliquots of the same ejaculates by colloid centrifugation to create SP-free sperm samples; the resuspended sperm pellets were treated with homologous or heterologous SP from high or low fertility bulls at 0%, 1% or 5% before freezing. After thawing, sperm quality was evaluated by computer-assisted sperm analysis and flow cytometry for membrane integrity, reactive oxygen species, chromatin structure, mitochondrial membrane potential and protein tyrosine phosphorylation. Data were analysed using Proc MIXED, SAS®. Post-hoc comparisons were adjusted for multiplicity using Tukey's method. The addition of SP resulted in significant differences in sperm quality, namely velocity class A, Velocity Straight Line (VSL), Velocity Average Path (VAP), Velocity Curved Line (VCL), Amplitude of Lateral Head Displacement (ALH), Hyperactive (HYP), reactive oxygen species (ROS) production and % DNA fragmentation index (DFI) (P<0.05 for each). Although adding 5% homologous SP from high fertility bulls was beneficial to sperm kinematics, 5% heterologous SP from high fertility bulls had a deleterious effect on chromatin integrity and on sperm velocity. In conclusion, adding SP may have either a beneficial effect or a deleterious effect depending on the individuals involved. It might be feasible to use this method to improve sperm quality in some circumstances.

Relationship between morphological abnormalities in commercial bull frozen semen doses and conception rate.

Commercial doses of frozen bull semen for artificial insemination may have a certain percentage of morphological defects, despite being subject to prior selection. The aims of this study were to determine the prevalence of morphological abnormalities in commercial doses (n = 55, r = 2) of dairy and beef bulls, from AI Centers and to determine the possible existence of differences between them, regarding the percentage of abnormal spermatozoa. At least 200 spermatozoa per sample were evaluated using Bengal Rose stain (3% m/v) and light microscopy (×1000 magnification). The mean percentage of abnormal sperm samples from dairy breeds was 7.19% ± 4.91% and from beef breeds was 15.83% ± 9.28%. Significant differences between biotypes were found in the proportion of abnormal spermatozoa, abnormal heads and abnormal midpieces; it could be due to different selection pressure. It was observed that the percentage of abnormal spermatozoa was not a good fertility level predictor for the commercial samples of frozen bovine semen used in this study. In both biotypes, the midpiece abnormalities were the most frequent, mainly its distal flexion (compensable defect). This could be as a result of the effects of freezing and thawing on spermatozoa.

A novel combination of silane-coated silica colloid with hybrid RNA extraction protocol and RNA enrichment for downstream applications of spermatozoal RNA.

Spermatozoa are specialised cells with low RNA content as compared to somatic cells. The suitable sperm RNA extraction and enrichment protocols for downstream applications are available for human, cattle, stallion and mouse but not for buffalo spermatozoa. Therefore, the present work was conducted to find out suitable colloidal solution for sperm purification and appropriate protocol for sperm RNA extraction and enrichment/amplification of RNA. For purification, we used PVP-coated silica colloidal solution (PVP-Si), silane-coated silica colloidal solution (Silane-Si) and iodixanol. Sperm recovery rate, total sperm motility and progressive sperm motility were significantly improved after separation by Silane-Si and iodixanol compared to PVA-Si method. The combined guanidinium thiocyanate-phenol-chloroform (GTPC) with silica matrix (SM)-based RNA extraction yielded more quantity of RNA in compared to individual method. The hybrid of SM and GTPC into a single protocol yielded 360-450 ng RNA from 30 million buffalo spermatozoa. For the first time, we adopted new way to enrich sperm RNA that increased the RNA concentration 4-5 times that was sufficient for downstream applications. The linear amplification of sperm RNA increased RNA concentration around 27-45 times. In summary, Silane-Si colloid for sperm separation, hybrid SM and GTPC protocol for sperm RNA extraction followed by enrichment or amplification of RNA was found suitable for high-throughput analyses of buffalo sperm RNA.

Effect of cryoprotectants and thawing temperatures on chicken sperm quality.

There is need for standardization of freezing-thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post-thaw motility and to analyse combined effect of the best permeating cryoprotectant (P-CPA) with one of four non-permeating cryoprotectants (N-CPA) on post-thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N-methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N-CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing-thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen-thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post-thaw motility. Moreover, ficoll addition to EG-based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N-CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.