161 CRYOPRESERVATION OF CORN SNAKE, ELAPHE GUTATTA, SEMEN

2009 ◽  
Vol 21 (1) ◽  
pp. 179 ◽  
Author(s):  
K. M. Mattson ◽  
A. T. DeVries ◽  
J. Krebs ◽  
N. M. Loskutoff
Keyword(s):  
Sperm Quality ◽  
Gradient Centrifugation ◽  
Egg Yolk ◽  
Genetic Management ◽  
Local Pressure ◽  
Chi Square ◽  
The Mean ◽  
Chi Square Analysis ◽  
Corn Snake

The purpose of this investigation was to develop a protocol for cryopreserving snake semen using the corn snake, Elaphe gutatta, as the model species. This experiment is part of a five year investigation where the influences of diluents, cryoprotectants, cooling and thawing rates on sperm survival were studied. This report presents one protocol found to be effective for cryopreserving corn snake semen as determined by post-thaw motility parameters in vitro. Semen was collected by applying pressure to the lower abdomen and continuing distally towards the cloaca to remove any feces or urates. The cloaca was washed using PBS, then a more local pressure was applied to each side of the vent to cause the hemipenes to evert and subsequently ejaculate. The semen (approximately 5 μL) was then collected using a sterile transfer pipette, placed in 120 μL Biladyl A containing 20% egg yolk (Minitube, 13502/0501), and analyzed for motility, rate of forward progression (RFP; 0–5), and concentration. The semen was further diluted at room temperature at 1:1 v/v with Biladyl A containing 20% egg yolk and 34% Glycerol (Sigma, G2025), yielding a final concentration of 17% Glycerol. The diluted semen was then loaded into 250-μL straws and slowly cooled for 1 hour. The straws were then placed 1 inch above a liquid nitrogen bath for ten minutes and finally plunged into the nitrogen where it remained frozen. The cryopreserved semen was thawed by placing the straws into a 50°C water bath for 8 s, then emptied into microcentrifuge tubes and the sperm were evaluated for motility and RFP. The mean motility of the fresh samples was 72.5% (66.4–77.7%). The mean post-thaw motility of sperm over six trials was 27.1% (17.8–50.2%). The mean RFP was 0.75 (0.5–1.0). The differences between fresh and post-thawed mean motilities were shown to be significant using a chi-square analysis (P < 0.0001). Density gradient centrifugation (DGC) was applied in one trial where the semen had an initial post-thaw motility of 50.2% with an RFP of 0.5. After the centrifugation treatment, the motility increased to 64.8% with an RFP of 3. The DGC media was composed of 400 μL 45% Percoll (Sigma, P4937) layered over 400 μL 90% Percoll. The density gradients were centrifuged at 700g for 30 min after which time the pellets were washed in 500 μL pre-warmed TL Hepes Solution (Lonza, 04-616F) and centrifuged at 300g for 10 min to remove the Percoll. The resulting sperm pellets were then resuspended in a small volume of the pre-warmed Hepes. Thus far, the protocol using 17% Glycerol in Biladyl A with 20% egg yolk has proven to be the most successful for cryopreserving corn snake semen. The use of DGC enhanced the number of usable sperm leaving sperm of higher motility and RFP possibly due to the absence of seminal plasma or cryoprotective agents that may detrimentally affect sperm quality. There are no known reports of the use of DCG with snake semen. Further studies are underway to improve these results and successfully use cryopreserved snake semen for artificial insemination and cryobanking for the long-term genetic management of endangered snake species.

scholarly journals 98CONCEPTION RATES USING BRAHMAN BULL SEMEN FROZEN IN MILK BASED EXTENDER CONTAINING EGG YOLK OR SOYBEAN LIPIDS; A FIELD STUDY IN A TROPICAL ENVIRONMENT

2004 ◽  
Vol 16 (2) ◽  
pp. 170 ◽  
Author(s):  
R. Gonzales ◽  
M. Rosales ◽  
F. Perea ◽  
J. Velarde ◽  
E. Soto ◽  
...  
Keyword(s):  
Egg Yolk ◽  
Pregnancy Rates ◽  
Glycerol Concentration ◽  
Forest Environment ◽  
Chi Square ◽  
Bull Semen ◽  
Frozen Semen ◽  
The Mean ◽  
Semen Extender ◽  
Chi Square Analysis

The objective of this study was to examine the substitution of soybean-origin phospholipids for egg yolk in Brahman bull semen extender. Semen was frozen in 3 different low-fat milk (1%) based extenders containing 10mgmL−1 of fructose and supplemented with: 8% of whole egg yolk (Extender 1, control), 8% rectified egg yolk (egg yolk granules were removed by double centrifugation at 3000g for 1h at 5°C; Extender 2), and 7.3mgmL−1 of phospholipids of soybean-origin containing 10% of phosphatidyl choline (Extender 3). All 3 extenders were supplemented with 1000IU of penicillin, 1mgmL−1 streptomycin and 150μgmL−1 lincomycin. The semen was collected by means of artificial vagina from 3 Brahman bulls, and AI was performed during the dry season between December and April in a tropical forest environment. The mean temperature for the region was 26–30°C, with mean rainfall of 900–1500mm/year and the relative humidity of 60–70%. Ejaculates with at least 60% motility were diluted in 2 steps as follows: in step 1, each ejaculate was split into 3 even parts and diluted at 26°C with each of the extenders containing no glycerol, and in step 2, 14% of glycerol was added in 15-minute intervals to a final glycerol concentration of 7%. Semen was aspirated into 0.5mL plastic straws (20×106 sperm/per straw), frozen 7cm above liquid nitrogen (LN2) for 8min, and then plunged into LN2. Straws were thawed in a water bath at 37°C for 30s. Each experiment was replicated 3 times (different collection days). Sperm viability was tested within artificial insemination trials. Results are based on the pregnancy rates of crossbreed Brahman cows determined by palpation 45 Days after AI and by calving rates. Data were compared by chi-square analysis. In Experiment I, a total of 157 cows were inseminated with semen collected from 3 different bulls (A, B and C) and frozen in 3 different extenders (1, 2 and 3; 3×3 factorial design). Bull A, Extender 1, 2 and 3 (n=19, 20 and 22); Bull B, Extender 1, 2 and 3 (n=20, 20 and 20) and Bull C, Extender 1, 2 and 3 (n=22, 15 and 24), respectively. Although semen from all 3 bulls frozen in Extenders 2 and 3 fostered numerically higher pregnancy rates (from 30% for Bull B and Extender 2 to 50% for Bull C and Extender 3) than in Extender 1 (from 23.5% for Bull C to 40% for Bull B), there were no differences (P&lt;0.05) between bulls with any of 3 extenders on the pregnancy rates. In Experiment II, a total of 117 cows were inseminated with semen collected from Bull B and frozen in Extender: 1 (n=37), 2 (n=48) and 3 (n=39). There were significantly higher (P&lt;0.05) calving rates for cows inseminated with semen frozen in Extender 2 and 3 (41.6% and 46.1%, respectively) than in Extender 1 (24.3%). It can be concluded that rectified egg yolk may improve viability of frozen semen, and that phospholipids of soybean origin can be successfully substituted for egg yolk in Brahman bull milk based semen extender. Supported by Bioniche Inc, Belleville, Ontario, Canada.

Surface culture of Steinernema sp. on two solid media and their pathogenicity against Galleria mellonella

2021 ◽  
Vol 95 ◽  
Author(s):  
C.I. Cortés-Martínez ◽  
A.I. Rodríguez-Hernández ◽  
M.R. López-Cuellar ◽  
N. Chavarría-Hernández
Keyword(s):  
Galleria Mellonella ◽  
Egg Yolk ◽  
Infective Juvenile ◽  
Surface Culture ◽  
Solid Media ◽  
Significant Difference ◽  
Bacterial Lawn ◽  
The Mean

Abstract The use of native entomopathogenic nematodes as biocontrol agents is a strategy to decrease the environmental impact of insecticides and achieve sustainable agriculture crops. In this study, the effect of the surface culture of Steinernema sp. JAP1 over two solid media at 23–27°C on infective juvenile (IJ) production and pathogenicity against Galleria mellonella larvae were investigated. First, the bacterial lawn on the surface of the media with egg yolk (P2) or chicken liver (Cl) were incubated in darkness at 30°C for 48 and 72 h, and 100 surface-sterilized IJs were added. Four harvests were conducted within the next 35 days and the mean accumulated production was superior on Cl (210 × 103 IJs) than on P2 (135 × 103 IJs), but the productivity decreased up to 10% when the incubation time of the bacterial lawn was of 72 h. The mean pathogenicity of in vitro- and in vivo-produced IJs were of 47–64% and 31%, respectively. It is worth noting that none of the two solid media had a statistically significant difference in IJ pathogenicity. Considering that the maximum multiplication factor of IJs on solid media was 2108 and that the pathogenicity against G. mellonella was outstanding, Steinernema sp. has a good potential for in vitro mass production.

191 INFLUENCE OF THE DIFFERENT TIME COMPONENTS BETWEEN FLUSHING AND TRANSFER ON PREGNANCY RATES OF FROZEN CATTLE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 203
Author(s):  
C. Ponsart ◽  
H. Quinton ◽  
A. Rohou ◽  
J. Kelhembo ◽  
G. Bourgoin ◽  
...  
Keyword(s):  
Pregnancy Rates ◽  
Chi Square ◽  
Frozen Embryos ◽  
Multivariate Logistic Model ◽  
Parity Number ◽  
Embryo Stage ◽  
Chi Square Analysis ◽  
Fresh Embryos ◽  
Donor Cows

Previous studies have shown that the time between flushing and freezing of bovine embryos can influence pregnancy rates (PRs) following embryo transfer (ET). The aim of this study was to determine which time components can influence ET results. Time components between flushing of a superovulated donor and freezing of the collected embryos were investigated under field conditions. Embryos were frozen in 1.5 M ethylene glycol (EG) for direct transfer. During January 2003, ET technicians (EmbryoTop, Rennes cedex, France) recorded systematically times corresponding to each step comprising the time spent in vitro (TIV) from 153 recovery sessions (RS) with freezing: end of flushing, beginning and end of search of embryos, start of equilibration in EG, beginning and end of straw loading, introduction to −7°C in the freezer, and seeding. Numbers of donor cows and ET technicians doing the freezing (n = 5) were noted for each RS. Embryo (stage, quality) and recipient (breed, parity) characteristics were also noted. A total of 548 frozen embryos were transferred and PRs were assessed. Variability of time components was investigated (Bourgoin et al. 2004 Reprod. Fertil. Dev. 16, 207). The influence of time components and other variation factors was tested on PRs (t-tests and chi-square analysis). The TIV averaged 210 ± 80 min and did not influence PR (≤4 h = 51.9% (n = 393) vs. >4 h = 55.5% (n = 155); P > 0.05), as well as duration of flushing (32 ± 8 min), interval between end of flushing and search (31 ± 27 min), duration of search (45 ± 25 min) and interval between end of search and beginning of freezing (101 ± 63 min). Only significant factors were kept for further analysis. The effects of recipient parity, number of donor cows per RS, and interval between introduction of straw to −7°C, and seeding were tested in a multivariate logistic model. PR varied strongly with parity of recipient (+25% in heifers vs. cows; P = 0.001). PRs were higher when the interval between straw introduction in the freezer and seeding lasted at least 5 min (2–4 min = 48.0% (n = 254) vs. 5–8 min = 57.1% (n = 294); P = 0.009). Time and operator effects were confounded. Overall PR results for the two technicians who used mostly 2–4 min intervals averaged 47% (operator values = 35.6, 48.9, and 54.5) whereas PRs were 54.9 and 60.5% for those waiting 5 min or more before inducing seeding (n = 2). PRs were higher when at least two donor cows were collected per RS (1 donor cow = 49% (n = 259) vs. ≥2 donor cows = 56.4% (n = 289); P = 0.003). This was not in agreement with previous observations in fresh embryos (Bourgoin et al. 2004). However, the number of donor cows strongly influenced the number of viable embryos per RS (1 donor cow = 11 ± 5 vs. ≥2 donor cows = 18 ± 8.5; P < 0.05) and could permit the choice of more embryos to be frozen. These results show that good PR may be achieved with a delay of several hours between flushing and freezing, when heifers are used as recipients. Moreover, confirmed from higher numbers of operators, these data show that it is better to wait at least 5 min to achieve equilibration of the embryo before seeding.

159 THE EFFECT OF DIFFERENT ZWITTERIONIC BUFFERS AND PBS ON IN VITRO DEVELOPMENT AND MORPHOLOGY OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 187
Author(s):  
J. De la Fuente ◽  
A. Gutiérrez-Adán ◽  
P. Beltrán Breña ◽  
S. S. Pérez-Garnelo ◽  
A. T. Palasz
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Apoptotic Cells ◽  
Bovine Embryos ◽  
Chi Square ◽  
Oh Groups ◽  
In Vitro Development ◽  
Chi Square Analysis ◽  
Vitro Development

It is assumed that, contrary to phosphate buffers, zwitterionic buffers are neutral. However, zwitterionic buffers containing hydroxymethyl or hydroxyethyl residues may interact with OH-groups in the media and produce formaldehyde (Shiraishi et al. 1993 Free Radic. Res. Commun. 19, 315-321). Also, it was shown that three zwitterionic buffers tested in this study interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167-175). Our objective was to evaluate the effect of the following buffers: TES (T), MOPS (M), HEPES (H) (pKa values at 20�C: 7.2-7.5), and PBS on in vitro development and morphology of bovine embryos. Zwitterionic buffers and PBS were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into four tubes. Collected oocytes (five replicates) from each tube were processed separately through the entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4 mg/mL BSA. Oocytes were matured in TCM-199 + 10% FCS and 10 ng/mL of epidermal growth factor and fertilized in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 �g/mL heparin with 1 � 106 spermatozoa/mL. After 24 h, oocytes-sperm co-incubation presumptive zygotes were cultured in SOFaa medium with 8 mg/mL BSA at 39�C under paraffin oil and 5% CO2 in humidified air. Cumulus-oocyte complexes and zygotes were held in designated buffers ?16 min before oocyte maturation, ~7 min after IVM and before IVF, and ~18 min after IVF and before culture. The total time of oocyte/embryo exposure to each buffer was ?41 min. Embryo development was recorded on Days 4, 7, 8, and 9. A total of ten, Day 8 blastocysts were taken randomly from each treatment and fixed in 4% paraformaldehyde for total and apoptotic cells counts, and five blastocysts from each replicate and treatment were frozen for later mRNA analysis. Apoptosis were determined by TUNEL, using commercial In situ Cell Death Detection Kit (Roche Diagnostic, SL, Barcelono, Spain). Embryo development among groups was compared by chi-square analysis. The cleavage rates were not different among the groups: PBS, 70.8%; H, 76.5%; M, 77.5% and T, 73.6%. The number of embryos that developed to d8 cells at Day 4 was higher in M, 36.2%, and PBS, 37.6%, than in H, 30.6%, and T, 29.7%, but was not significantly different. However, more (P < 0.05) blastocysts developed at Days 7, 8, and 9 in H and M than in PBS and T groups (21.9% and 22.9% vs. 16.9% and 14.9%, respectively). No difference was found between groups in total cell number (98.8 � 7, PBS; 111.8 � 11.9, M; 106.8 � 12.9, H; and 104.3 � 9.7, T) and the number of apoptotic cells (9.2 � 1.0, P; 9.2 � 0.8, M; 12.9 � 1.8, H; and 9.7 � 0.9, T). Based on the results of this study, we conclude that within our protocol choice of buffer may affect embryo developmental rates but not morphology.

122 TIMED ARTIFICIAL INSEMINATION IN WOOD BISON USING FROZEN-THAWED SEMEN

2016 ◽  
Vol 28 (2) ◽  
pp. 191 ◽  
Author(s):  
G. P. Adams ◽  
S. X. Yang ◽  
J. M. Palomino ◽  
M. Anzar
Keyword(s):  
Animal Health ◽  
Egg Yolk ◽  
Ovulation Rate ◽  
Chi Square ◽  
Semen Cryopreservation ◽  
Wood Bison ◽  
The Mean ◽  
Intravaginal Device ◽  
Synchronization Procedure

Recent progress with methods to control ovulation and semen cryopreservation in Wood Bison was the impetus to test the feasibility of timed AI to facilitate reclamation of this threatened species. A 2 × 2 design was used to compare the efficacy of 2 ovulation synchronization techniques and 2 semen cryopreservation protocols. Female Wood Bison were assigned randomly to 2 groups (n = 24/group) in which ovarian synchronization was induced by ultrasound-guided ablation of follicles >5 mm or intramuscular treatment with 2.5 mg of estradiol 17B + 50 mg of progesterone (E+P) in canola oil. A progesterone-releasing intravaginal device (PRID) was placed at the time of follicle ablation (for 5 days) or E+P treatment (for 8 days) in the respective groups. A luteolytic dose of prostaglandin was given at the time of PRID removal, and 2500 IU of hCG was given IM 3 days later. Bison were inseminated 24 and 36 h after hCG treatment using frozen-thawed semen. The semen was collected by electro-ejaculation from 4 Wood Bison bulls, pooled, and divided into aliquots diluted in either egg-yolk extender (EY) or cholesterol-loaded cyclodextrin extender (CLC). Half the bison in each synchronization group were inseminated with either EY- or CLC-extended semen. Bison were examined by ultrasonography every 12 h beginning on the day of hCG treatment for 3 days or until ovulation was detected, whichever occurred first. Pregnancy diagnosis was made by ultrasonography 34–36 days after insemination. Two bison were excluded during the experiment because of handling difficulty; therefore, the total number of bison used was 46. Ovulation rate and interval to ovulation were compared between synchronization groups by chi-square and t-test, respectively. Pregnancy rates were compared among groups by 2-way ANOVA after transforming data to arcsin. The ovulation rate was not different between synchronization groups [combined mean, 37/46 (80%)], nor was the degree of synchrony, as assessed by the residuals (variation from the mean) in the respective groups. However, the diameter (mean ± standard error of the mean) of the dominant follicle at the time of hCG treatment was smaller in the follicle ablation group than in the E+P group (10.5 ± 0.6 v. 13.9 ± 0.6; P < 0.04), and the interval from hCG treatment to ovulation tended to be longer (35.3 ± 1.6 v. 31.8 ± 1.3 h; P ≤ 0.10). Pregnancy rate was not affected by synchronization procedure, but pregnancy was detected only in the EY-inseminated group (9/23 v. 0/23; P < 0.01). Despite that post-thaw sperm motility was similar for EY and CLC semen (41.7 ± 2.9 and 44.6 ± 3.3%; respectively), CLC-treated semen failed to impregnate bison in vivo. We concluded that synchronization and timed insemination with frozen-thawed semen is feasible in Wood Bison. Of the 23 bison inseminated with EY-extended semen, 21 ovulated (91%), and of those that ovulated 9 became pregnant (43%). Both synchronization schemes were effective, but the ablation protocol may be improved by an additional day between ablation and hCG treatment. We thank Vetoquinol Canada and Merck Animal Health for providing hormone treatments.

212 EFFECT OF CONSECUTIVE SUPERSTIMULATORY TREATMENT-INDUCED FOLLICULAR WAVE SYNCHRONIZATION TO OPTIMIZE OOCYTE RETRIEVAL AND EMBRYO PRODUCTION BY OVUM PICKUP AND IN VITRO FERTILIZATION IN COWS

2011 ◽  
Vol 23 (1) ◽  
pp. 205
Author(s):  
K. Imai ◽  
M. Ohtake ◽  
Y. Aikawa ◽  
S. Sugimura ◽  
M. Hirayama ◽  
...  
Keyword(s):  
Development Program ◽  
Oocyte Retrieval ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Follicular Wave ◽  
The Third ◽  
The Mean ◽  
Student’S T

We previously reported that superstimulatory (SS) treatment-induced follicular wave synchronization after ovum pickup (OPU) was effective in enhancing the quality of obtained oocytes and blastocysts derived from in vitro maturation (IVM) and fertilization (IVF; Imai et al. 2010 Reprod. Fertil. Dev. 22, 296). The present study was designed to examine the efficiency of embryo production by 4 sessions of OPU-IVF using a series of the SS treatment-induced follicular wave synchronizations. For the SS protocols, 3 consecutive SS (3CSS) and 2 separated SS (2SSS) were used. In the 3CSS group, the first OPU was performed on random days of the oestrous cycle (Day 0) and all follicles larger than 2 mm in diameter were aspirated. On Day 5, follicles larger than 8 mm in diameter were aspirated and a CIDR (InterAg, Hamilton, New Zealand) was inserted. The cows then received 20 armour units of FSH (Kawasaki-Seiyaku, Kawasaki, Japan) in twice-daily decreasing doses by IM injection from Day 7 to 10. Cloprostenol (PGF; 0.75 mg, Fujita-Pharm, Tokyo, Japan) was administered on the morning of Day 9. The second OPU was performed 48 h after PGF administration on Day 11; the CIDR was removed from the cows just before OPU. After the second OPU, donors were treated consecutively with the SS protocol mentioned above for the third and fourth OPU sessions. In the 2SSS group, donors received 2 sets of the SS treatment mentioned above, with an interval of 11 days between the second and the third OPU session. Four OPU sessions were performed every 11 days on all cows. In this study, 8 Japanese Black cows were divided into the 3CSS and 2SSS groups, and the treatment for each group was reversed after a 65-day interval as crossover trials. After OPU, Grade 1 and 2 oocytes were used for IVM and IVF, and putative zygotes were cultured as described by (Imai et al. 2006 J. Reprod. Dev. 52, S19–S29 suppl.). A part of the zygotes were cultured in a micro-well system. Data were analysed by Student’s t-test and chi-square test. There were differences (P < 0.05) in the mean (±SD) number of follicles, collected oocytes, and cultured oocytes in the 3CSS (35.0 ± 8.6 and 24.4 ± 11.2, respectively) and 2SSS (30.8 ± 10.5 and 20.2 ± 9.0, respectively) groups. There were no differences in mean percentage of blastocyst formation and Grade 1 blastocyst rates between the 3CSS (38.5 and 55.8%, respectively) and 2SSS (34.8 and 54.8%, respectively) groups. However, the mean number of blastocysts produced per OPU session was significantly (P < 0.05) higher in the 3CSS group (8.1 ± 6.3) compared with the 2SSS group (5.8 ± 4.4). These results indicate that a series of 3 consecutive SS treatments had greater efficiency in producing OPU-IVF embryos. This work was supported in part by the Research and Development Program for New Bio-industry Initiatives.

scholarly journals 266IN VITRO-DERIVED EMBRYO PRODUCTION WITH SEXED AND UNSEXED SEMEN FROM DIFFERENT BULLS

2004 ◽  
Vol 16 (2) ◽  
pp. 253 ◽  
Author(s):  
L. Ferré ◽  
C. Ohlrichs ◽  
D. Faber
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Calf Serum ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Sexed Semen ◽  
Chi Square Analysis

The production of pre-sex-selected calves by in vitro fertilization (IVF), using sexed semen, does show some benefits due to the small quantity of sperms needed for the process as compared to other reproductive technologies. The objective of this study was to determine differences among bulls and sperm concentrations in embryo development with sexed and unsexed semen. Follicles ranging from 2 to 6mm in diameter were aspirated from slaughterhouse ovaries. COC were selected and matured in groups of maximum of 30 in 1.8mL of TCM-199, supplemented with 10% fetal calf serum, 0.01UmL−1 bFSH, 0.01UmL−1 bLH and 10μLmL−1 penicillin-streptomycin for 24h at 38.5°C. Fertilization (Day 0) was carried out in micro-drops (50μL) with TALP-FERT medium containing PHE (3μgmL−1 penicillamine, 11μgmL−1 hypotaurine and 0.18μgmL−1 epinephrine), 10μLmL−1 non-essential amino acid and 2μgmL−1 heparin. Frozen/thawed sexed (female) and non-sexed sperms from five bulls were selected in a discontinuous percoll gradient. Sperm concentration was 1×106 for non-sexed semen and 1×106 or 2×106 for sexed semen. After 18–20h, presumptive zygotes were denuded and cultured in groups of 10 in 50-μL micro-drops of SOF citrate with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700) under paraffin oil in a 5% O2, 5% CO2, 90% N2 atmosphere with high humidity. On Day 7, blastocysts (BL) were morphologically evaluated and recorded. Results are shown in Table 1. Data was compared by chi-square analysis. Sexed frozen bovine sperm can be used successfully in IVF systems. More research needs to be done to optimize and standardize bovine in vitro fertilization with sexed semen. Table 1 Results of comparisons between bulls, sperm concentrations, cleavage and embryo development

Flow cytometric and microscopic evaluation of post-thawed ram semen cryopreserved in chemically defined home-made or commercial extenders

2015 ◽  
Vol 55 (4) ◽  
pp. 551 ◽  
Author(s):  
M. Emamverdi ◽  
M. Zhandi ◽  
A. Zare Shahneh ◽  
M. Sharafi ◽  
A. Akhlaghi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Soybean Lecithin ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Mitochondrial Activity ◽  
Flow Cytometric ◽  
Microscopic Evaluation ◽  
Open Question ◽  
Ram Sperm

The present study was designed to determine the effect of three different extenders on ram sperm quality during a freeze–thawing procedure using flow cytometric and microscopic evaluations. Several in vitro qualitative analyses of post-thawed sperm parameters including motility and velocity parameters, plasma membrane functionality, total abnormality, capacitation status, acrosome integrity, mitochondrial activity and apoptosis features were considered. In the breeding season, seven ejaculates from each Zandi ram were collected routinely twice a week. Following semen collection, samples were pooled and equally divided into three aliquots. Each aliquot was diluted and frozen with one of the following extenders: (1) Tris-based extender containing 1.5% (w/v) soybean lecithin (TSL), as a chemically defined extender, (2) Bioxcell, a commercial soybean lecithin-based extender, and (3) Tris-based extender containing 20% (v/v) egg yolk (TEY). The results of the present study indicated no differences in total [TSL (55.8 ± 2.02%) vs TEY (50.2 ± 2.02%; P < 0.05)] and progressive motility of spermatozoa [TSL (26.2 ± 1.36%) vs Bioxcell (22.4 ± 1.36%; P < 0.05)]. Semen freezing by means of TSL resulted in a higher percentage of live spermatozoa (39.42 ± 1.81%) compared with TEY (29.17 ± 1.81%; P < 0.05), and a higher percentage of functional plasma membrane (50.8 ± 192%) compared with TEY (44 ± 1.92%) and Bioxcell (38.8 ± 1.92%; P < 0.05). The effect of extenders on sperm capacitation status showed that the percentage of post-thawed capacitated spermatozoa was higher in TEY (61.9 ± 1.48%) compared with that in TSL (56.6 ± 1.48%; P < 0.05). The evaluation of post-thawed spermatozoa indicated that the percentage of live spermatozoa with active mitochondria was higher in TSL (53.05 ± 2.31%) compared with Bioxcell (45.92 ± 2.31; P < 0.05) and the percentage of intact acrosome spermatozoa was higher in TSL (84.55 ± 2.51%) compared with TEY (74.91 ± 2.51%; P < 0.05). The use of TSL and Bioxcell extenders reduced the percentage of apoptotic spermatozoa (40.82 ± 2.07% and 42.22 ± 2.07%, respectively), compared with TEY (51.34 ± 2.07%; P < 0.05). Post-thawing dead spermatozoa were increased when semen was frozen by Bioxcell (25.69 ± 1.28%). The results of this study showed that TSL extender may provide stabile milieu and conditions for ram sperm cryopreservation compared with Bioxcell and TEY extenders. Whether TSL extender can improve the artificial insemination results remains, however, an open question.

scholarly journals Effect of whole and clarified egg yolk based extenders on post-thaw semen quality in bulls.

2021 ◽  
Author(s):  
Prosper Kamusasa ◽  
Eddington Gororo ◽  
Fungayi Primrose Chatiza
Keyword(s):  
Semen Quality ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Normal Morphology ◽  
Semen Cryopreservation ◽  
Bull Semen ◽  
Inclusion Level ◽  
Whole Egg

Abstract This study was conducted to evaluate the comparative cryoprotective effects of whole egg yolk and clarified egg yolk on post thaw sperm quality parameters and to determine the optimum clarified egg yolk inclusion level (10-20%) in semen extenders for Mashona bull semen cryopreservation. It was shown that there was a significant decrease in sperm quality variables following cryopreservation. Semen quality increased with the concentration of clarified egg yolk, indicating a positive relationship between egg yolk LDL concentration and maintenance of in vitro sperm quality. The 20% clarified egg yolk (CEY20) extender treatment gave post-thaw motility, viability and normal morphology values which were comparable to the control (20% whole egg yolk, WEY20). The 10% clarified egg yolk concentration gave the least post-thaw quality values and the greatest proportion of defective spermatozoa. This experiment found no advantage of replacing whole egg yolk with up to 15% clarified egg yolk in Mashona bull semen cryopreservation. However, 20% clarified and 20% whole egg yolk performed similarly in the maintenance of post-thaw sperm motility, viability and normal morphology.

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