Tyrode's Albumin Lactate Pyruvate Research Articles

Effect of tyrode albumin lactate pyruvate and modified Krebs Ringers broth media on in vitro capacitation of HD-K75 boar spermatozoa at different period of incubation.

In vitro capacitation allows for a greater understanding of the mechanisms underlying fertilization and the development of improved reproductive techniques for improving fertility rates in porcine. Tyrodes albumin lactate pyruvate (TALP) and modified Krebs Ringers Broth (m-KRB) are two medias that are commonly used in research experiments to induce capacitation in boar spermatozoa (Cañón-Beltrán etal., Theriogenology, 198, 2023 and 231; Oberlender etal., Archivos de Medicina Veterinaria, 44, 2012 and 201; Sahoo etal., International Journal of Biological Macromolecules, 241, 2023 and 124502). Moreover, understanding the morphological and functional changes in boar spermatozoa at different hours of capacitation periods might aid in the development of novel techniques for improving sperm quality and increasing the litter size. This study was carried out to investigate the effect of Tyrode albumin lactate pyruvate and modified Krebs Ringers Broth media on invitro capacitation of HD-K75 boar spermatozoa at three different periods of incubation. A total of 24 ejaculate from four clinically healthy, 10-12 months aged HD-K75 boars, maintained at ICAR-All India Coordinated Research Project (AICRP) on pig were selected. Semen was collected by 'Simple fist' method using a portable dummy. The semen samples having 200 mL volume, 103 × 106 spermatozoa/ml concentration and 70% initial motility were selected and split into two parts and suspended in TALP and m-KRB media, respectively, and incubated for 5 h at 37°C. Seminal parameters viz. sperm viability, plasma membrane integrity and acrosomal integrity were estimated in the samples at 0, 3 and 5 h of incubation. This study revealed that there was significant variation between media in live acrosome-reacted (p < .05) and HOST-reacted (p < .01) spermatozoa, while between capacitation periods significant (p < .01) variation was observed in hyperactivated spermatozoa, live acrosome-reacted spermatozoa, HOST-reacted spermatozoa, FITC-labelled PSA, extracellular protein and sperm cholesterol. Non-significant variation was observed in total phospholipid. TALP showed overall better consequence on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. From this study, it could be concluded that both TALP and m-KRB media were virtuous to induce capacitation in HD-K75 boar spermatozoa. TALP media, however, had a better effect on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. Out of the three different periods, 3 h capacitation period resulted in significantly (p < .01) higher incidence of sperm viability, plasma membrane and acrosomal integrity in HD-K75 boar spermatozoa.

Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.

Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol=120.9±2.9 vs. NC=76.91±6.9µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol=1.15±0.02 vs. NC=0.77±0.03µm) (straightness: bicarbonate and polyvinyl-alcohol=0.76±0.01 vs. NC=0.87±0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.

Efficient and repeatable in vitro fertilization in rabbits

Rabbits constitute an interesting model to understand gamete interaction and test novel Artificial Reproductive Techniques, but in vitro fertilization (IVF) is particularly problematic in this species. We have conducted a series of experiments to develop a consistent IVF technique. Initially, we checked viability, acrosome integrity, capacitation and motility in ejaculated sperm purified by a density gradient and incubated at different times in three different media: Tyrode's Albumin Lactate Pyruvate (TALP), human tubal fluid (HTF), and Brackett and Oliphant (BO). Total and progressive motility at 10–24 h and linearity from 3 h onwards was significantly higher in BO medium compared to TALP and HTF. Subsequently, cumulus-oocyte complexes (COCs) collected 10 h after induction of ovulation were incubated with sperm in TALP, HTF or BO for 18 h with or without performing sperm pre-incubation for 6 h. Pronuclear formation rate at 18 h was significantly higher in BO compared to other media (∼84 % vs. 17–22 %) and was not improved by pre-incubation. As COCs recovery rate was low at 10 h after induction of ovulation, COCs were collected at 12 h and co-incubated with sperm in BO. Pronuclear formation rate was similar than those obtained in COCs collected at 10 h (∼85 %), and when embryos were allowed to develop in vitro, the protocol yielded high cleavage and blastocyst rates (91 and 59 %, respectively). In conclusion, ejaculated rabbit sperm purified in a density gradient fertilize efficiently COCs collected at 12 h in BO medium.

Effect of FertTalp addition to stallion semen on kinetic parameters and production of intracellular hydrogen peroxide after thawing

Motility stimulants increase sperm energy production and their addition after semen thawing will increase motility (Stephens et al., J. Equine Vet. Sci. 2013;33:615-621), and stimulate metabolic pathways responsible for sperm capacitation (Guasti et al., Anim. Reprod. Sci. 2017;179:27-34). Semen of 12 Mangalarga Marchador stallions was cryopreserved with addition of different concentrations of cholesterol-loaded cyclodextrin (CLC): G1 (control – without cholesterol), G2 (1mg of cholesterol), G3 (1.5mg of cholesterol) and G4 (2mg of cholesterol). To run the experiment, two straws from each stallion per experimental group were thawed and divided into two aliquots of 400µL. This was supplemented with 100µL of Fert Talp, a Tyrode's albumin lactate pyruvate medium with (FT) or without (No-FT) heparin, a motility stimulant. This subdivided the four groups into eight. Sperm kinetics were performed analyzed by CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®). Sperm longevity was analyzed by using a thermoresistance test at 37°C (TTR). Statistical analysis was done by ANOVA F-test. Complementary to sperm kinetics, the production of intracellular hydrogen peroxide was estimated by flow cytometry using Dichlorodihydrofluorescein diacetate (DCFDA®). Results were classified as: Q1 [Plasma membrane (PM) injured without intracellular hydrogen peroxide]; Q2 (PM injured with intracellularhydrogen peroxide); Q3 (intact MP with intracellular hydrogen peroxide); Q4 (intact PM without intracellular hydrogen peroxide) and Tukey test was performed for statistical analysis. Immediately after thawing, there was no difference between FT and No-FT (p>0.05). After incubation at 37°C, the addition of FT showed higher values for RAP (percentage of rapid sperm, %), VAP (average path velocity, μm/s) and LIN (linearity of the curvilinear trajectory, %) after 80 minutes of incubation and also superior rates of RAP, VSL (straight line velocity, μm/s), VAP and LIN after 120 minutes of incubation (p<0.05), demonstrating satisfactory results both with regard to sperm kinetics and longevity. It can be concluded that the use of FT was beneficial for sperm velocity. These findings suggest that the addition of this motility stimulant after thawing may improve fertility because sperm velocity – VCL (curvilinear velocity, μm/s), VSL and VAP are the most reliable parameters to assess sperm quality (Verstegen et al. Theriogenology, 2002;57:149-179). Significant differences were also observed for Q4 (intact PM, without hydrogen peroxide), where the G2 FT group demonstrated superiority when compared to all groups not supplemented with FT (p>0.05), providing satisfactory plasmatic membrane results against oxidative stress.

Influence of hypoxanthine concentrations on capacitation, acrosome reaction and In vitro fertilization in Ram Spermatozoa

The aim of the study was to evaluate the effect of hypoxanthine concentrations and incubation time on motility, hyperactivity (HA) and acrosome reaction (AR) in ram sperm in vitro. Semen samples (n= 40) were collected using artificial vagina from 3 mature Barki rams. Freshly ejaculated spermatozoa were pooled and subjected to swim up technique in modified sperm Tyrode's albumin lactate pyruvate (S-TALP) medium supplemented with different concentrations of hypoxanthine (5, 10, 15, 20 mg/ml). Motility, hyperactivity percentage and acrosomal status were recorded at 0, 1, 2, 3 and 4 h post-incubation. They were examined for ability to fertilize oocyte in vitro. The results showed that hypoxanthine concentrations and incubation time for 2 h did not significantly affect the motility. However, increase in incubation time for >2 h significantly (P<0.05) decreased the motility of spermatozoa as compared to control. Treatment of spermatozoa with 20 mg/ml hypoxanthine significant (P<0.05) increased in HA after 2 h of incubation compared to control (5.2±0.3 vs. 1.0± 0.1%). Significant (P<0.05) increase in total AR was associated to the increase in concentration of hypoxanthine with maximum at 20 mg/ml at 3h of incubation (82.0±2.2%). The fertilization rate of oocytes treated with 20 mg/ml hypoxanthine was higher as compared with control (42.8 vs. 26.0%). In conclusion, treatment of ram spermatozoa with 20 mg/ml hypoxanthine for 2 to 3 h was considered the best concentration for in vitro fertilization in sheep.

Cryoprotectant-free vitrification of llama spermatozoa: cryoloop vs sphere method, warmed rapidly or ultra-rapidly

Our objective was to determine the effect of vitrification using both cryoloop and sphere methods, combined with two warming protocols, on the percentage of motility, membrane integrity and DNA fragmentation/condensation of llama spermatozoa. Raw semen (n = 5; r = 3) was incubated with collagenase (0.1 % in Tyrode's Albumin Lactate Pyruvate, TALP, solution), centrifuged and diluted in TALP with or without 5% dimethylformamide (DMF). Two spermatozoa concentrations were used: 20 × 106 sperm/mL (cryoloops) and 5 × 106 sperm/mL (spheres). Two warming procedures were evaluated: a rapid method (30 s at 37 °C) and an ultra-rapid method (7 s at 75 °C, followed by 30 s at 37 °C). Percentages of sperm total motility, viability, membrane function, chromatin condensation and chromatin fragmentation were evaluated before and after vitrification and analyzed using Friedman’s test. The only seminal parameters maintained following vitrification were: 1) chromatin condensation, in all evaluated protocols and 2) DNA integrity, with the spheres method using both warming protocols without cryoprotectants and warming rapidly with cryoprotectants. Spermatozoa vitrification is a simple and inexpensive cryopreservation method that was able to preserve llama sperm chromatin condensation and integrity, especially using the spheres method without cryoprotectants combined with warming at 37 °C for 30 s. However it failed to preserve spermatozoa motility, membranes and viability.

Use of Androcoll-ETM to Separate Frozen-Thawed Llama Sperm From Seminal Plasma and Diluent.

It is not easy to separate frozen-thawed South American camelid sperm from seminal plasma (SP) and diluents to be used for in vitro embryo production. The objective of this study was to evaluate Androcoll-E™ (AE) efficiency to separate llama sperm from SP and freezing extender in frozen-thawed semen. A total of 22 ejaculates from five Lama glama males were collected using electroejaculation. After performing semen analysis (sperm motility, concentration, viability, membrane function, and acrosome integrity), samples were cryopreserved with a diluent containing lactose, ethylenediaminetetraacetic acid (EDTA), egg yolk, and 7% dimethylformamide. After thawing, samples were divided in aliquots, one of which was used as a control and the others processed by AE. Experiment 1 (12 ejaculates): 100 μl of frozen-thawed semen was placed on top of 1,000 μl AE column and centrifuged at 800 g for 10 min. Experiment 2 (10 ejaculates): two samples of 100 μl of frozen-thawed semen were placed on two columns of 500 μl AE each, and both were centrifuged at 800 g for 10 and 20 min, respectively. Pellets were resuspended in Tyrode's albumin lactate pyruvate (TALP) medium, and sperm parameters were evaluated. A significant decrease in all sperm parameters was observed in thawed samples compared to raw semen. AE allowed the separation of frozen-thawed sperm from SP and freezing extender independently from the height of the column used and time of centrifugation assayed. Although no significant differences were found between AE columns, higher sperm recovery was observed with 500 μl of AE coupled with 20 min of centrifugation. Despite the significant decrease observed in sperm motility in AE samples, no changes in sperm viability, membrane function, and acrosome integrity were observed when comparing control thawed semen with the sperm recovered after AE (p > 0.05). The use of AE columns, either 500 or 1,000 μl, allows the separation of frozen-thawed llama sperm from SP and freezing extender, preserving the viability, membrane function, and acrosome integrity. Of the protocols studied, 800 g centrifugation during 20 min using a 500 μl column of AE would be the method of choice to process frozen-thawed llama semen destined for reproductive biotechnologies.

Acute mild heat stress alters gene expression in testes and reduces sperm quality in mice

Heat stress is a major concern in animal reproduction, as testicular temperature must be 3–5 °C below body core temperature for production of motile and fertile sperm in mammals. Although recent studies concluded that increased temperature per se was the underlying pathophysiology of testicular impairment, more studies are required to better understand the mechanisms. Therefore, our objective was to investigate the impacts of mild acute heat stress on sperm and testes, and based on mRNA, elucidate involvement of StAR, Trp53 and Trp53-dependent intrinsic and extrinsic apoptotic pathways in pathophysiology of testicular heat stress. Forty-eight C57 BCL6 elite male mice were equally allocated into six groups, anesthetized and the distal third of their body immersed in a water-bath at 40 or 30 °C (heat treatment and control, respectively) for 20 min. Intervals from heat exposure (Day 0) to euthanasia were: 8 and 24 h and 7, 14 and 21 d (plus a control group at 14 d). The epididymides were excised, minced and placed in Tyrode albumin lactate pyruvate hepes (TALPH) at 37 °C for 15 min to recover sperm. Based on computer assisted sperm analysis (CASA), heat treatment reduced total and progressive motility ∼40% (P < 0.05) on Days 14 and 21. Furthermore, percentage morphologically normal sperm was significantly decreased on Day 7, with greater reductions on Days 14 and 21, mostly due to increased midpiece defects. Acrosome integrity (FITC PSA) was decreased ∼35% at 8 h (P < 0.05) and reached a nadir on Day 14. There were decreases (P < 0.05) in seminiferous tubule diameter and testicular weight (relative to body weight) on Day 14. Testicular RNA was extracted, reverse-transcribed and cDNA used for PCR. Expression of genes Hspa1b (Hsp70) and Gpx1 had 7- and 10-fold increases (P < 0.001 for each) at 8 and 24 h, respectively, with Hspa1b remaining upregulated at 24 h, whereas StAR peaked at Day 14 (15-fold, P < 0.0001) and had returned to baseline on Day 21. Both Trp53 and Casp8 were upregulated (P < 0.05) on Day 14, whereas Bcl-2 was decreased (P < 0.05) on Days 7 and 14. In conclusion, acute mild heat stress severely reduced sperm quality and based on mRNA, there was upregulation of chaperone and antioxidant systems and Trp53-dependent intrinsic and extrinsic apoptotic pathways, with deleterious effects on sperm, spermatocytes and spermatids. These findings provided insights into the pathophysiology of heat stress and should contribute to development of evidence-based approaches to mitigate effects of testicular heating.

22 The zona pellucida is required for normal development of invitro-produced cat embryos

Limited information have been reported regarding to the role of the zona pellucida during embryo development in the domestic cat. In the present research we compared invitro and invivo development of cat embryos generated with versus without the zona pellucida. Embryos were produced by IVF and cultured up to blastocyst stage accordingly to experimental group: 1) with intact zona pellucida (ZI) and 2) without zona pellucida (ZF). Ovaries of domestic cats were collected by ovariohysterectomy and cumulus-oocyte complexes (COCs) were recovered by slicing. For IVM, COCs were cultured in supplemented medium-199 for 26-28h in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C. For IVF, 1.5-2.5×106 epididymal spermatozoa/mL were incubated with 20-30 COCs in supplemented Tyrode's albumin lactate pyruvate medium for 18h in a 5% CO2 atmosphere at 38.5°C. The zona pellucida of the presumed zygotes was removed by 2-4min of incubation in 2mgmL−1 pronase. In ZI and ZF groups, embryo culture was done in 4-well dishes; in the ZF group the well of the well system was used. The IVC was done in supplemented synthetic oviductal fluid medium, in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C for 8 days. The frequencies of cleavage and development to the morula and blastocyst stages were determined. The relative expression of the pluripotency markers OCT4, SOX2, and NANOG was evaluated by RT-qPCR in the blastocysts using the standard curve method. The SDHA gene was used as internal control. Additionally, the invivo development was evaluated. For this, cat recipients were synchronized with 200IU equine chorionic gonadotrophin followed by 100IU human chorionic gonadotrophin 4 days later, and embryo transfers (ET) were made by mid-ventral laparotomy. The Wilcoxon nonparametric test was used to evaluate the developmental competence and the gene expression analysis. Nine and six replicates were performed in the ZI and ZF groups, respectively. No differences were observed between the ZI and ZF groups in the cleavage rate: 155/239 (64.9%) and 116/177 (65.5%), morulae rate: 115/155 (74.2%) and 68/116 (58.6%), and blastocysts rate: 51/155 (32.9%) and 36/116 (31.0), respectively (P&amp;gt;0.05). No differences were observed in the expression of OCT4 (P&amp;gt;0.05). However, the expression of SOX2 and NANOG was lower in blastocysts from the ZF group than in those from the ZI group (P&amp;lt;0.05). Finally, three ET were done at the blastocyst stage in both groups. In the ZI group, 8-16 Day 7 blastocysts were transferred per cat, two pregnancies were obtained with one gestational sac in each cat (2/3, 66.6%), and one live kitten was born at Day 64 of pregnancy (1/3, 33.3%). In the ZF group, 7-14 were transferred per cat, and no pregnancies were obtained after ET. In conclusion, in the domestic cat, zona pellucida removal at the presumed zygote stage did not affect invitro development to blastocyst but affect negatively the expression of SOX2 and NANOG. However, the low survival after ET of ZI embryos is a possible indicator that embryo quality might be affecting survival rates. Nonetheless, these results add credence to previous ET studies that provide indirect evidence of a crucial role of the zona pellucida for successful implantation of cat embryos.

83 Validation of propidium iodide dye for live-dead staining of bovine blastocysts: Preliminary results

A widely used criterion routine laboratories utilise for the selection and classification of oocytes and embryos is morphological appearance. In order to make an objective validation of produced embryos, it is necessary to have a method that can help to distinguish viable cells. One simple and easy-to-perform method is the live-dead staining protocol. Usually, ethidium homodimer (EthD) is used to stain the dead cells, in conjunction with Hoechst 33342 for total cell numbers (Stinshoff et al. 2011). However, EthD is a harmful, toxic, and expensive staining dye. Therefore, the aim of this study was to evaluate propidium iodide (PI) as a viable alternative to EthD. To assess the suitability of PI, groups of 20 cumulus-oocyte complexes were collected from abattoir-derived bovine ovaries and matured in tissue culture medium containing bovine serum albumin and equine chorionic gonadotropin/human chorionic gonadotropin for 24h with 5% CO2. The cumulus-oocyte complexes were then fertilised in Fert-TALP (Tyrode's albumin lactate pyruvate) for 19h with 5% CO2 and finally cultured (n=968) in SOFaa (synthetic oviductal fluid with amino acids) with 5% CO2 and 5% O2 with all steps being performed at 39°C. The development rate on Day 8 was on average 25.4%. Expanded Day 8 blastocysts were then utilised to validate the two different staining protocols. Expanded blastocysts were collected from culture drops and washed in PBS containing polyvinyl alcohol. The blastocysts were then stained with the selective dye and finally counterstained to determine live and dead cells. Four alternative parameters were evaluated: (A) PI 1:5 for 15min, (B) PI 1:10 for 15min, (C) PI 1:5 for 5min, and (D) EthD 1:10 for 15min (control). Counterstaining with Hoechst 33342 was performed for 3min in all groups tested. A minimum number of 10 replicate stainings ((A) n=13, (B) n=13, (C) n=12, (D) n=10) was carried out. The evaluation was performed under a fluorescence microscope (Nikon Eclipse Ci) with the NIS-Elements imaging software (Nikon). The statistical analysis was assessed using R. All data were analysed for normal distribution using a Shapiro-Wilk test. Evaluation of the data was made through the application of t-tests. The total cell numbers observed were in the known range for expanded blastocysts: (A) 106.3±15.3, (B) 109.5±18.8, (C) 119.3±15.0, and (D) 113.1±21.8. The number of dead cells did not differ significantly from the control: (A) 13.3±7.7, (B) 8.5±5.0, (C) 11.8±8.7, and (D) 9.3±2.3. The study indicates that PI could be used as an adequate alternative for EthD. Furthermore, it is possible that the concentration or the incubation time could be reduced to generate similar results to EthD. Further replicates are currently being examined in order to corroborate these results. We gratefully acknowledge our colleague M. Vopel.

146 Tributyltin chloride exposure alters ejaculated bull sperm function and embryo development

Male exposure to environmental toxicants can disrupt spermatogenesis and impair sperm function. However, the consequences of environmentally relevant levels of toxicants to ejaculated mammalian spermatozoa on sperm function and male fertility are not well studied. Tributyltin chloride (TBT) is an organotin with historical use as an antifouling agent in paints and is a contaminant of soil and groundwater in the United States. Tributyltin chloride is an endocrine disruptor, is detectable in human cord blood, and has negative effects on female reproduction. We hypothesised that TBT could affect sperm function and thereby affect male fertility. To test our hypothesis, we exposed frozen-thawed bull sperm to environmentally relevant doses of TBT (0, 0.1, 1.0, 10, and 100nM) for 90min and then measured sperm motility parameters, fertilisation, and embryo development by IVF. Briefly, frozen-thawed sperm from two bulls were isolated through a 45:90 Percoll gradient, pooled, and then maintained in noncapacitating conditions at 37°C in Tyrode's albumin lactate pyruvate medium devoid of bovine serum albumin and HCO3 − for 90min. Vehicle control (VC) samples consisted of 0.1% MeOH. Sperm motility kinematics were objectively measured after the addition of treatment and every 30min thereafter using computer-aided sperm analysis (IVOS System, Hamilton Thorne). Five replicates were evaluated, and differences in motility kinematics were analysed by analysis of variance using SAS statistical software (SAS Institute Inc.). Sperm treated with 100nM TBT displayed decreased total motility (88 vs. 79%), progressive motility (80 vs. 70%), curvilinear velocity (100 vs. 88 µ/s), and beat-cross frequency (38 vs. 34Hz) over 90min compared with the VC samples (P&amp;lt;0.05). No differences (P&amp;gt;0.05) were detected among any other treatments. Following 90min of exposure to TBT 100nM, sperm were washed twice by centrifugation and re-extended in fertilisation medium. Abattoir-derived bovine oocytes were fertilised with 100nM TBT and VC-exposed sperm. Embryo cleavage and 8- to 16-cell embryos were quantified at 48 and 72h, respectively, in three replicates, and results were assessed using chi-square analysis. Embryos fertilised by TBT-exposed sperm had reduced cleavage to 2-cell (80 vs. 62%) and 8- to 16-cell morulae stages (56 vs. 24%, respectively; P&amp;lt;0.05). In summary, although sperm kinematics were decreased in TBT-exposed sperm, gross motility parameters remained within acceptable ranges for IVF, suggesting that sperm motility alone is not a sufficient measure of sperm function or indicator of male fertility. In conclusion, ejaculated bull sperm exposed to environmentally relevant levels of TBT for 90min had reduced sperm motility parameters, impaired sperm function, and reduced embryo development potential. Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award number T32HD087166. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

37 In vitro maturation and fertilization in white-tailed deer (Odocoileus virginianus) oocytes vitrified with trehalose or sucrose

White-tailed deer (WTD) invitro embryo production is not well documented, due to the seasonality of the wildlife species and available hunting permits. The objective of the present study was to conduct IVM and IVF using WTD oocytes vitrified with trehalose (TH) or sucrose (SC). A total of 121 immature oocytes were obtained from ovaries (n=18) using the slicing technique from hunter-slaughtered deer, not more than 2h after death. The WTD oocytes were vitrified using a solid surface vitrification technique in two different groups: 1) TH (n=60) and 2) SC (n=61). Oocyte warming (OW) was done using four concentrations (OW1 1 M, OW2 0.5 M, OW3 0.25 M, and OW4 0 M) in the oocytes after thawing. Then, a sample was used to evaluate viability for TH (n=5) and SC (n=5) (MTT stain (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 0.5mgmL−1) and nuclear status (NS) for TH (n=4) and SC (n=5) with 4′,6-diamidino-2-phenylindole (1μgmL−1), defining the stage as germinal vesicle (GV), MI, and unable to evaluate (NE). The remaining oocytes (n=88) were used for IVM for 36h in tissue culture medium-199 supplemented with human menopausal hormone (75 UImL−1) and epidermal growth factor (10ngmL−1). The evaluation of NS was defined (GV, MI considered immature and MII matured). The IVF (n=15) took place in Tyrode's albumin lactate pyruvate media supplemented with heparin (10μgmL−1), penicillamine (0.075mgmL−1), hypotaurine (10M), epinephrine (1μM), and bovine serum albumin fraction V (0.4%), with a final semen concentration of 3×106 spermmL−1. Frozen straws of conventional WTD semen were used; the straws were split into 3 parts, with one fraction thawed and capacitated using “swim-up” technique. After 24h of incubation at 38.5°C with 5% CO2, and humidified air, nuclear evaluation was made as fertilized (F), not fertilized (NF), or NE. A Fisher exact test was used (SAS, 9.0 version for Windows), α=0.05. After warming, for the TH group (n=5), viability was 60% and nuclear status (n=4) was 50% GV, 50% MI, and 0% NE. For the SC group (n=5), viability was 40% and nuclear status (n=5) was 60% GV, 20% MI, and 20% NE. After 36h IVM, NS evaluation in the TH group (n=38) was 66% GV, 24% MI, 0% MII, and 10% NE; for the SC group (n=50), 84% GV, 10% MI, 2% MII, and 4% NE, not a statistically significant difference (P&amp;gt;0.05). For IVF after 24h, the NS evaluation in the TH treatment (n=10) was 0% F, 60% NF, and 40% NE versus the SC treatment (n=5) with 20% F, 40% NF, and 40% NE, not a statistically significant difference (P&amp;gt;0.05). A statistically significant difference (P&amp;gt;0.05) was not found between the TH and SC groups with regard to post-thaw viability, IVM, and IVF. Future research with larger numbers of immature or mature oocytes is suggested for further evaluation of both TH and SC for WTD oocyte vitrification for use with invitro-production techniques as a model for other endangered cervid species.

143 Stimulated glycolysis is able to maintain ATP levels and motility of bull spermatozoa submitted to mitochondrial depolarisation

Studies have reported the importance of mitochondria in sperm metabolism. However, for some species, glycolysis appears to be as essential as oxidative phosphorylation in sperm physiology. On the other hand, these mechanisms have not been fully elucidated for bovine spermatozoa. Therefore, the aim of this study was to evaluate the role of mitochondria and glycolysis in ATP synthesis and sperm kinetics of bovine spermatozoa. For this purpose, sperm from seven bovine epididymides (n=7) was collected and diluted to a concentration of 100×106 spermatozoamL−1 in Tyrode's albumin lactate pyruvate medium. Then, each sample was divided into 10 aliquots and evaluated in a 2×5 factorial design, with the first factor being the presence or absence of glucose (5mM) to stimulate glycolysis and the second factor being treatment with the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 0, 0.1, 0.3, 1, and 3 µM) to deplete mitochondrial ATP. Sperm samples were subjected to measurements of ATP levels using a luminescence technique (CellTiter-Glokit, Promega), with ATP levels measured in duplicate. In addition, sperm samples were subjected to computerised analysis of total and progressive motilities (ISAS, Proiser). Statistical analysis was performed using SAS (SAS Institute Inc.), where the interaction between the factors was analysed using PROC GLM and the comparison between groups was performed using means analysis of variance (least significant difference test). It was considered significant at 5%. Adenosine triphosphate was lower at FCCP concentrations of 0.3 µM (180.3±31.9nM), 1 µM (220.2±40.4nM), and 3 µM (272.3±70.4nM) than at 0 µM (control; 448.6±63.7nM) and 0.1 µM (422.4±41.5nM) in the absence of glucose. However, in the groups treated with FCCP supplemented with glucose, ATP concentrations did not differ among the groups (0 µM: 577.2±70.4 nM; 0.1 µM: 610.8±57.8 nM; 0.3 µM: 606.2±64.2 nM; 1 µM: 670.9±61.9 nM; 3 µM: 696.1±68.5nM). Additionally, total motility was lower in FCCP-treated groups without glucose supplementation. On the other hand, total motility increased in the groups treated with 0.3, 0.1, 1, and 3 µM FCCP supplemented with glucose. A similar effect was verified for progressive motility. Based on these results, we can suggest that glucose supplementation is able to maintain ATP levels and motility in bull sperm undergoing FCCP-induced mitochondrial depolarisation.

Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines.

Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation? Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling. In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process. Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 μM), Gö6983 (PKC inhibitor, 10 μM), PD98059 (ERK-1/2 inhibitor, 30 μM), H89 and KT (PKA inhibitors, 50 μM and 100 nM, respectively), KH7 (sAC inhibitor, 10 μM), BAPTA-AM (intracellular Ca2+ chelator, 50 μM), EGTA (10 μM) and Probenecid (MRPs general inhibitor, 500 μM). In addition, assays for binding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such as heparin (60 μg/ml) and bicarbonate (40 mM). Straws of frozen bovine semen (20-25 × 106 spermatozoa/ml) were kindly provided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry, flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independent assays with bull samples of proven fertility were carried. In the present study, we elucidate the molecular events induced by extracellular cAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzyme increased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitation also depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad range of responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa with high mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001). None. This is an in vitro study performed with bovine cryopreserved spermatozoa. Studies in other species and with fresh samples are needed to extrapolate these data. These findings strongly suggest an important role of extracellular cAMP in the regulation of the signalling pathways involved in the acquisition of bull sperm fertilizing capability. The data presented here indicate that not only a rise, but also a regulation of cAMP levels is necessary to ensure sperm fertilizing ability. Thus, exclusion of the nucleotide to the extracellular space might be essential to guarantee the achievement of a cAMP tone, needed for all capacitation-associated events to take place. Moreover, the ability of cAMP to trigger such broad and complex signalling events allows us to hypothesize that cAMP is a self-produced autocrine/paracrine factor, and supports the emerging paradigm that spermatozoa do not compete but, in fact, communicate with each other. A precise understanding of the functional competence of mammalian spermatozoa is essential to generate clinical advances in the treatment of infertility and the development of novel contraceptive strategies. This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas [PIP0 496 to S.P.-M.], Agencia Nacional de Promoción Científica y Tecológica [PICT 2012-1195 and PICT2014-2325 to S.P.-M., and PICT 2013-2050 to C.D.], Boehringer Ingelheim Funds, and the Swedish Farmers Foundation [SLF-H13300339 to J.M.]. The authors declare there are no conflicts of interests.

Role of the Calcium-Sensing Receptor (CaSR) in bovine gametes and during in vitro fertilization

Calcium Sensing Receptor (CaSR) is a G-protein coupled receptor which senses extracellular calcium and activates diverse intracellular pathways. The objective of our work was to demonstrate the presence of CaSR in bovine gametes and its possible role in fertilization and embryo development. The location of CaSR was demonstrated by immunofluorescence in bovine gametes; additionally bovine sperm were incubated with 5, 10 and 15 μM of the specific CaSR inhibitor NPS2143 in a Tyrode's Albumin Lactate Pyruvate medium (4 h). Sperm viability was maintained for all concentrations tested while total motility decreased significantly at 10 and 15 μM. Addition of 15 μM of NPS2143 during oocyte in vitro maturation did not alter the maturation rate. When NPS2143 (15 μM) was added to the fertilization medium during sperm-oocyte co-incubation the cleavage, morula and blastocyst rates remained unchanged. To confirm if 15 μM of NPS2143 exerted any effect on embryo development, NPS2143 was added to the embryo culture medium. Cleavage rates remained unchanged when 15 μM of NPS2143 was added to the culture medium (79.1 ± 6.8 vs. 73.7 ± 5.3; mean % ± SEM; p > 0.05, control vs. inhibitor). By contrast, development to the morula (46.6 ± 7.3 vs. 24.3 ± 4.3; mean % ± SEM; p < 0.05) and blastocyst stages (29.9 ± 9.0 vs. 9.9 ± 3.6; mean % ± SEM; p < 0.05) decreased (control vs. inhibitor). Our results demonstrate a key role of CaSR on sperm motility and embryo development but not on oocyte maturation or fertilization in the bovine species.

Semen Collection and Spermatozoa Characteristics in the Kea Parrot (Nestor notabilis).

We describe the seminal characteristics of the kea parrot (Nestor notabilis), an endangered species endemic to the South Island of New Zealand. Semen was collected in the full breeding season from 6 birds in the collection of an amateur aviculturist. The manual massage technique was used. A total of 25 ejaculates was collected and evaluated for volume, degree of contamination, and spermatozoa concentration; motility and kinetic parameters were assessed on diluted samples (modified Tyrode's Albumin Lactate Pyruvate, pH 8.2, temperature 37.5°C) with a computer-aided sperm analyzer. Four ejaculates were not analyzed because of an excessively high degree of contamination. Semen color ranged from transparent or turbid yellow to whitish. The geometric mean of spermatozoa number/ejaculate was 765.9 ± 2084.7 × 106. Total and progressive motility were 71.7% ± 20.0% and 59.8% ± 22.0%, respectively. Great variability was observed both among birds and among different ejaculates of the same subject. The seminal characteristics of kea are worth further investigation, with the aim of relating semen quality to fertility and defining a minimum inseminating dose for breeding purposes. A deeper knowledge of male reproductive biology also can increase the success of breeding programs and enable the use of the kea as a model species for other more threatened species, such as the kaka ( Nestor meridionalis ) and the kakapo (Strigops habroptila).

Study of TALP and TRIS citrate medium on caprine sperm capacitation and subsequent in vitro embryo production.

The aim of the present investigation was to compare Tyrode's albumin lactate pyruvate (TALP) medium and TRIS citrate medium for capacitation of caprine sperm. In experiment 1 capacitation was assessed by chlortetracycline assay and in experiment 2 with in vitro fertilization and embryo development. In experiment 2, cumulus oocyte complexes (COCs) recovered by slicing the caprine ovaries were matured in maturation medium for 27 h in humidified atmosphere at 38.5°C with 5% CO2. After 27 h of culture a total of 2480 in vitro matured oocytes were selected and randomly divided into two groups. Group 1 (n=1124) matured oocytes were fertilized by the spermatozoa capacitated in TALP medium and in group 2 (n=1356) matured oocytes were fertilized by the spermatozoa capacitated in TRIS citrate medium. The results of experiment 1 indicated a comparatively more number of sperms with Chlortetracycline (CTC) Pattern B in TRIS citrate than TALP medium (55.32 ± 0.91% vs 47.96 ± 0.20%). In experiment 2, the cleavage rate and blastocyst production were higher following capacitation of spermatozoa in TRIS citrate than TALP medium. In conclusion, TRIS citrate can be used as an alternative and effective media for sperm capacitation to get higher cleavage rate and blastocyst production in goat.

Effects of penicillamine, hypotaurine, and epinephrine on motility, hyperactivity, acrosome reaction of fresh ram sperm

Objective: To determine the effects of different concentrations of penicillamine, hypotaurine, and epinephrine (PHE) as well as incubation time on motility, hyperactivity and acrosome reaction (AR) of ram sperm in vitro. Methods: Freshly ejaculated spermatozoa from three ram were collected, pooled and subjected to swim up technique in modified sperm Tyrode's albumin lactate pyruvate medium supplemented with different concentrations of PHE (10, 20, 30, 40, 50, 75 and 100 mM/mL). Then best concentrations were compared and examined for motility, hyperactivity and AR. Results: A high concentrations of PHE (30, 40, 50, 75 and 100mM/mL) showed a significant increase in motility when compared to control immediately after dilution and exist for the first and second hour of incubation period. However, when longer incubation time were used, a significant (P

Effect of L-arginine treatment on motility, hyperactivity, acrosome reaction of ejaculated ram spermatozoa

The aim of this study was to evaluate the effect of in vitro treatment of ejaculated ram spermatozoa with different concentrations of L-arginine at various incubation times on motility, hyperactivity (HA) and acrosome reaction. Freshly ejaculated spermatozoa collected from three rams were pooled and subjected to the swim up technique in modified sperm Tyrode's albumin lactate pyruvate (S-TALP) medium supplemented with different concentrations of Larginine (0.01, 0.02, 0.03,0.04 and 0.05 mM) at 30, 60,90 and 120 min of incubation. Following sperm incubation, the following parameters were examined: motility, hyperactivity (HA) and acrosome reaction (AR). The results showed that irrespective of the concentration, incubation of ram spermatozoa with Larginine for 30min did not significantly affect the motility. However, increase the time of incubation for more than 30 min significantly decreased (P < 0.05) the motility of the spermatozoa as compared to the control. The lowest motility was recorded when spermatozoa were incubated with 0.05 mM L-arginine for 120 min. Treatment of ram spermatozoa with 0.05 mM Larginine resulted in a significant (P < 0.05) increase in HA% immediately after dilution compared to the control. A significant (P < 0.05) increase in total AR% was concomitant to the increase in the concentration of L-arginine with highest AR achieved at 0.04 mM and 90 min incubation. However, increasing the time of incubation to 120 min significantly decreased (P < 0.05) the percentage of spermatozoa with total AR compared to the other incubation times at 0.02, 0.04, and 0.05 mM L-arginine. In conclusion, under our experimental conditions treatment of ejaculated ram spermatozoa with 0.04 mM of L-arginine for 90 min was considered the best concentration of L-arginine to be used for in vitro induction of acrosome reaction.

Effect of melatonin on maturation capacity and fertilization of Nili-Ravi buffalo (Bubalus bubalis) oocytes.

This study evaluated the effect of melatonin supplementation of in vitro maturation media on in vitro maturation (IVM) and in vitro fertilization (IVF) rate of buffalo oocytes. Cumulus oocytes complexes (COCs) were aspirated from follicles of 2-8 mm diameter. In experiment I, COCs were matured in IVM medium supplemented with 0 (control), 250, 500, and 1000 μM melatonin for 22-24 hours in CO2 incubator at 38.5°C with 5% CO2 and at 95% relative humidity. The maturation rate did not differ in media supplemented with melatonin at 250 μM, 500 μM, 1000 μM and control (0 μM). In experiment II, the matured oocytes were fertilized in 50 μl droplets of Tyrode’s Albumin Lactate Pyruvate (TALP) medium having 10 ug/ml heparin for sperm (2 million/ml) capacitation. The fertilization droplets were then kept for incubation at 5% CO2, 39°C and at 95% relative humidity for 18 hours. The fertilization rate was assessed by sperm penetration and pronuclear formation. Fertilization rate was improved when maturation medium was supplemented with 250 μM melatonin compared to control. In conclusion, melatonin supplementation to serum free maturation media at 250 μM improved the fertilization rate of buffalo oocytes.