216 IN VITRO OOCYTE MATURATION, FERTILIZATION, AND CULTURE AFTER LAPAROSCOPIC OVUM PICK-UP IN AN ENDANGERED GAZELLE (GAZELLA DAMA MHORR)

2006 ◽  
Vol 18 (2) ◽  
pp. 216 ◽  
Author(s):  
F. Berlinguer ◽  
S. Succu ◽  
A. del Olmo ◽  
R. Gonzalez ◽  
J. J. Garde ◽  
...  
Keyword(s):  
Sperm Concentration ◽  
Egg Yolk ◽  
Mature Female ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Ministry Of Education ◽  
Hormonal Stimulation ◽  
Vitro Fertilization ◽  
Genetic Value

The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks. Studies have been carried out on endangered Mohor gazelle sperm cryopreservation (Garde et al. 2003 Biol. Reprod. 69, 602-611), but there are no studies on oocytes in this species. The purpose of this work was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC. The study was conducted using six reproductively mature female Mohor gazelles from the breeding herd at the Estacion Experimental de Zonas Aridas. Animals were synchronized by insertion of controlled progesterone internal drug release (CIDR) devices for 14 days and removal of the devices on the day of ovum pickup (OPU). Follicular growth was stimulated by a total of 5.28 mg of oFSH (Ovagen, ICP, Auckland, New Zealand) given in four equal doses every 12 h. OPUs were performed (Berlinguer et al. 2004 Theriogenology 61, 1477-1486) on Day 15 from the beginning of treatment, and follicles were aspirated with a syringe and a 25G needle using TCM199-HEPES with 50 �g/mL streptomycin, 50 IU/mL penicillin, 0.1% polyvinyl alcohol, and 15 IU/mL heparin. Degenerated oocytes and those with expanded cumulus were removed. Oocytes were cultured in TCM-199 plus 10% FCS, 10 �g/mL ovine FSH/LH, 1 �g/mL estradiol, and 0.1 mg/mL glutamine at 38.5�C under 5% CO2/air and maximum humidity. Spermatozoa were cryopreserved in Tes-Tris with 5% egg yolk and 6% glycerol, and selected by swim-up in SOF medium. After 24 h sperm-oocyte coincubation (sperm concentration: 1 � 106/mL) in SOF with 2% estrus sheep serum under 5% CO2 5% O2 90% N2, presumptive zygotes were transferred to SOF with 0.4% BSA and amino acids under 5% CO2, 5% O2 90% N2 and cultured for 4 days. Oocytes and embryos were stained with Hoechst 33342 and propidium iodide (1 �g/mL each) and visualized under a fluorescence microscope. A total of 35 oocytes were recovered from 56 punctured follicles (62.5%). This recovery rate was similar to those in wildlife in earlier reports, but more studies are needed to improve hormonal stimulation and oocyte harvesting. Out of 29 cumulus-oocyte complexes matured in vitro, 3.5% were found at GV and 6.9% at MI; 20.7% were degenerated and 68.9% had advanced to MII. Fertilization and cleavage rates were 40% and 30%, respectively, of matured oocytes. Out of eight zygotes, six showed cleavage (ranging from 2 to 8 cells). None of the developing embryos progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more trials will help to improve IVMFC, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by OPU from FSH-stimulated endangered gazelles. This work was supported by the Spanish Ministry of Education and Science (REN 2003-11587) and Acciones Integradas (HI20030336).

284 COMPARATIVE FERTILITY OF FRESHLY-COLLECTED VERSUS FROZEN - THAWED SPERMATOZOA FOR IN VITRO FERTILIZATION IN THE FISHING CAT (PRIONAILURUS VIVERRINUS)

2006 ◽  
Vol 18 (2) ◽  
pp. 249
Author(s):  
G. Magarey ◽  
J. Herrick ◽  
K. Thiangtum ◽  
W. Tunwattana ◽  
W. Swanson
Keyword(s):  
Sperm Motility ◽  
Culture Medium ◽  
Culture Media ◽  
Egg Yolk ◽  
Ovarian Follicles ◽  
Blastocyst Stage ◽  
Motile Sperm ◽  
Vitro Fertilization ◽  
International Transport

Wild populations of fishing cats (Prionailurus viverrinus) in Southeast Asia are in decline, primarily due to habitat loss. Because the fishing cat population in North American zoos is small (n = 69) and inbred (F = 0.17) with relatively low genetic variation (86%), infusion of new founder genes from Asia is a conservation priority. Importation of cryopreserved semen for use with IVF and ET may offer one alternative to the international transport of living animals. In this study, our objectives were to (1) compare motility longevity of fresh vs. frozen-thawed fishing cat spermatozoa in two culture media, (2) evaluate ovarian responses to exogenous gonadotropins, and (3) assess development of IVF embryos produced with fresh vs. frozen-thawed spermatozoa. Raw semen was collected via electroejaculation from male fishing cats (n = 4), divided into groups, and washed. Two sperm pellets were resuspended in either Ham's F10 medium (HF10; with 5% FBS) or our feline optimized culture medium (FOCM; with 0.4% BSA); another pellet was diluted in TEST egg yolk, cooled to 5�C over 3 h, glycerated (4%), and cryopreserved in straws over LN2 vapor. Frozen sperm samples were thawed, washed, and diluted in either HF10 or FOCM. Fresh and frozen-thawed sperm motility (percent motile, rate of forward progress) in each medium (10 � 106 motile sperm/mL) was assessed (at 0, 1, 3, and 6 h) in microdrops under oil during culture (38�C; 6% CO2 in air). Female fishing cats (n = 10) were treated with exogenous gonadotropins (150 IU eCG, 100 IU hCG, 85-h interval) and ovarian follicles were aspirated laparoscopically. Recovered oocytes were inseminated with fresh (2 � 105 motile sperm/mL) or frozen-thawed (5 � 105 motile sperm/mL) spermatozoa in FOCM microdrops; resulting embryos were either cryopreserved or cultured in FOCM (with 5% FBS added at 72 h post-insemination) for 7 days. Sperm motility over time did not differ (P > 0.05) between media for either fresh or frozen-thawed samples; however, across media, frozen-thawed sperm motility was lower (P < 0.05) and declined faster (P < 0.05) compared to fresh spermatozoa. Females produced an average (�SEM) of 9.8 � 2.9 mature ovarian follicles, allowing recovery of 7.3 � 2.6 high-quality oocytes per female. Oocyte cleavage percentage at 42 h p.i. was lower (P < 0.05) with frozen-thawed spermatozoa (38%, 11/29) compared to freshly collected spermatozoa (68%, 17/25). Overall, 35% (6/17) of cultured embryos developed to blastocysts with no difference (P > 0.05) between embryos produced with frozen-thawed (4/11) vs. fresh (2/6) spermatozoa. Although fishing cat sperm motility and fertility appear compromised after cryopreservation, our results demonstrate the ability of frozen-thawed spermatozoa to produce IVF embryos that are capable of developing to blastocyst stage in vitro. This work was supported by (NIH RR015388).

scholarly journals 289EFFECT OF FERTILIZATION TIME OF PIG OOCYTES MATURED IN-VITRO BY BOAR SPERM STORED AT 4°C

2004 ◽  
Vol 16 (2) ◽  
pp. 264
Author(s):  
Y.J. Yi ◽  
M.Y. Kim ◽  
Y.J. Chang ◽  
D.I. Jin ◽  
C.S. Park
Keyword(s):  
In Vitro Fertilization ◽  
Room Temperature ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
Penicillin G ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Boar Sperm ◽  
Sperm Penetration

The use of boar sperm stored at 4°C may be a useful tool for in vitro production of pig embryos. Therefore, this study was undertaken to investigate the effect of fertilization time of pig oocytes matured in-vitro by boar sperm. The sperm-rich fraction (30–60mL) was slowly cooled to room temperature (20–23°C) by 2h after collection. Semen was transferred into 15-mL tubes, centrifuged at room temperature for 10min at 800g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5mL of the LEN (11.0g lactose hydrate, 20mL egg yolk, 0.05g N-acetyl-D-glucosamine and 100mL distilled water) diluent to provide 1.0×109 spermmL−1 at room temperature. The resuspended semen was cooled in a refrigerator to 4°C. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Oocytes were inseminated with boar sperm stored at 4°C for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9h in 500μL TBM fertilization media with 1×106mL−1 sperm concentration. Thereafter, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture of 6, 48 and 144h, fixed and stained for the evaluation of fertilization parameters and developmental ability. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times of 6 and 9h than in those of 1 and 3h. The percentage of polyspermic oocytes was highest in fertilization time of 9h compared with other incubation times. The rates of cleaved oocytes were higher in the fertilization times of 6 and 9h (85.0 and 84.6%) compared with those of 1 and 3h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time of 6h (33.6%) than in that of 1, 3 and 9h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9±3.3, 27.6±2.7, 26.3±2.2 and 24.4±1.8 in the fertilization times of 6, 9, 3 and 1h, respectively. In conclusion, we found out that boar sperm stored at 4°C could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend the coincubation time of 6h in 500μL TBM fertilization medium with 1×106mL−1 sperm concentration for in vitro fertilization of pig oocytes matured in vitro.

Porcine oocyte preincubation in oviductal fluid flush before in vitro fertilization in the presence of oviductal epithelial cells improves monospermic zygote production

Zygote ◽  
2021 ◽  
pp. 1-8
Author(s):  
Ribrio Ivan Tavares Pereira Batista ◽  
Lucia N. Moro ◽  
Emilie Corbin ◽  
Carmen Alminana ◽  
Joanna Maria Gonçalves Souza-Fabjan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Penetration Rate ◽  
Sperm Concentration ◽  
Major Effect ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Vitro Fertilization ◽  
Porcine Oocyte ◽  
Low Efficiency

Summary The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.

120 Theophylline: the Key for Bulls with Low In Vitro Blastocyst Rates?

2018 ◽  
Vol 30 (1) ◽  
pp. 199
Author(s):  
S. M. Bernal-Ulloa ◽  
S. E. Ulbrich
Keyword(s):  
In Vitro Fertilization ◽  
Sperm Concentration ◽  
Underlying Mechanism ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Penetration Ability ◽  
Bull Sperm ◽  
Low Performance ◽  
Pde Inhibition

Although many efforts have been made to improve the sperm normal penetration ability for in vitro embryo production (IVP), sperm capacitation in bovine remains a bottleneck for in vitro fertilization (IVF). The variable responses of sperm to the different supplements in IVF medium makes the process suboptimal for every bull. Theophylline, a methylxanthine, has recently been reported to enhance and maintain in vitro blastocyst production due to prolonged motility and longevity of bovine sperm (Kang et al. 2015 J. Reprod. Dev. 61, 99-105). Here, we evaluated the effects of theophylline supplementation during IVF on developmental rates of bulls with known low and average blastocyst production under standard conditions. A total of 1498 cumulus–oocyte complexes were obtained by slicing from bovine ovaries. Cumulus-oocyte complexes were submitted to in vitro maturation (IVM) for 24 h. In vitro fertilization was performed for 19 h with (2.5 mM) or without theophylline using frozen–thawed sperm from 5 different bulls at a final sperm concentration of to 2 × 106 cells mL−1. Two of the bulls had a previous report of low blastocyst rates (A, B) and 3 showed an average blastocyst production (C, D, E). After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. R software (https://www.r-project.org/) was used to evaluate cleavage and blastocyst rates using a two-sample t-test. The supplementation of theophylline significantly increased blastocyst rates for bulls A, B, and E (Table 1; P < 0.05). The blastocyst rates did not differ for bull C and D with or without the use of theophylline (Table 1; P > 0.05). These preliminary results show that in vitro blastocyst production for low performance bulls can be improved by theophylline supplementation during IVF. The underlying mechanism probably involves phosphodiesterase (PDE) inhibition to increase intra-sperm/oocyte cAMP levels improving oocyte-sperm interaction. Table 1.Total oocytes and mean (± SEM) cleavage and blastocyst rates of bull sperm treated or not with theophylline The authors thank Susanne Meese and Ulrich Witschi from Swissgenetics for the cooperation and donation of frozen bull sperm.

scholarly journals 252 COMPARISON OF TWO SPERM SEPARATION PRODUCTS FOR USE IN BOVINE IVF

2005 ◽  
Vol 17 (2) ◽  
pp. 276 ◽  
Author(s):  
J. Pryor ◽  
S. Romo ◽  
D.D. Varner ◽  
K. Hinrichs ◽  
C.R. Looney
Keyword(s):  
Sperm Concentration ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Before And After ◽  
Chi Square Test ◽  
Group 2 ◽  
Live Sperm ◽  
Sperm Separation

In commercial bovine in vitro fertilization (IVF) companies, there is a continuous need to improve results. Efforts to maximize in vitro embryo production have included modifications in the use of sperm separation gradients. The development of commercially available sperm centrifugation gradients represents a new possibility of increasing the number of viable sperm that can be obtained from low concentration (fresh or frozen, sexed or unsexed) semen samples in order to improve the efficiency of the IVF system to make embryo production as efficient as possible. The objective of this study was to compare two different separation gradients, as follows: Group 1: Percoll (Sigma, St. Louis, MO, USA), in 45% and 90% gradients; Group 2: EquiPure (Nidacon, Gathenburg, Sweden), in top and bottom layers. Before and after separation, sperm were evaluated at 200× magnification for total motility, and then stained to assess viability at 400× with fast-green/eosin stain (Sigma). Sperm separation was performed using frozen/thawed semen from one bull. Semen was separated by centrifugation at 200g for 30 min in both density gradients. Results obtained from Groups 1 and 2 were compared by chi-square test. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 92 × 106, vs. 159 × 106 sperm/mL for EquiPure; P < 0.05) but resulted in higher motility (60% vs. 39%, respectively; P < 0.05) of separated sperm. Rates of live sperm cells were not significantly different between groups (69.5% vs. 70%, respectively; P > 0.1). These results indicate that the commercial separation medium EquiPure may be associated with higher sperm concentration levels but with lowered sperm motility when compared to Percoll for bovine sperm separation. However, Equipure provided similar percentages of live sperm when compared to Percoll, which is currently used in our laboratory.

Growth and viability of human blastocysts in vitro

2000 ◽  
Vol 8 (3) ◽  
pp. 241-287 ◽  
Author(s):  
GM Jones
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Early Cleavage ◽  
Uterine Endometrium ◽  
Stage Of Development ◽  
Uterine Lavage

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.

Calibration of sperm concentration for in vitro fertilization in a mouse reprotoxicity model

2019 ◽  
Vol 55 ◽  
pp. 58-61 ◽  
Author(s):  
Hanne Skovsgaard Pedersen ◽  
Ying Liu ◽  
Leslie Foldager ◽  
Henrik Callesen ◽  
Knud Larsen ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Sperm Concentration ◽  
Vitro Fertilization

scholarly journals L-OPU in Goat and Sheep—Different Variants of the Oocyte Recovery Method

Animals ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 658
Author(s):  
Jarosław Wieczorek ◽  
Jurij Koseniuk ◽  
Maria Skrzyszowska ◽  
Mirosław Cegła
Keyword(s):  
In Vitro Maturation ◽  
Hormonal Stimulation ◽  
Recovery Method ◽  
Vitro Fertilization ◽  
Minimal Invasiveness ◽  
Animal Reproduction ◽  
Oocyte Recovery ◽  
Maturation In Vitro ◽  
Invasive Techniques

The laparoscopic method of recovering oocytes in goats and sheep is one of the minimally invasive methods used in the biotechnology of animal reproduction. It allows for good quality oocytes that are suitable for in vitro maturation and fertilization to be recovered. The limitation of using the laparoscopic ovum pick-up (L-OPU) method in goat and sheep is its changing effectiveness and the lack of repeatability of results, as well as the varying effectiveness of different variants of the method. Therefore, it is necessary to develop effective non-invasive techniques allowing for multiple good quality oocyte recovery that would be suitable for in vitro maturation and fertilization. In this study, four different L-OPU variants were described in goats and sheep. Various techniques of recovering oocytes were discussed, including the techniques of conducting the operation, various tools for recovering oocytes, and different plans of hormonal stimulation. Recovery rates were 35% (Variant I), 57% (Variant II), 72% (Variant III), and 67% (Variant IV). After evaluation, 94% (both Variant I and II), 93% (Variant III), and 84% (Variant IV) of the oocytes were qualified for in vitro maturation. The results of the study show that the proposed technique of laparoscopic recovery of oocytes allows a sufficient number of ovarian cells suitable for in vitro culture to be obtained and as a consequence it makes them useful in in vitro maturation/in vitro fertilization (IVM/IVF) programs or cloning. The method allows for a fast and effective conduct of the operation in a living donor with minimal invasiveness while preserving the excellent condition of animals.

scholarly journals 253 COMPARISON OF THREE DIFFERENT IVF PROTOCOLS FOR BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 277
Author(s):  
S. Romo ◽  
J. Pryor ◽  
D.D. Varner ◽  
K. Hinrichs ◽  
C.R. Looney
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Sperm Concentration ◽  
Vitro Fertilization ◽  
Group 3 ◽  
Group 2 ◽  
The Given ◽  
Sperm Separation ◽  
Group 1

Recently, the development of commercially available defined media and sperm centrifugation gradients has offered new possibilities for increasing the efficiency of commercial in vitro fertilization (IVF) systems. The objective of this study was to compare three different IVF protocols using two different separation gradients, two fertilization media, and two embryo culture media, as follows: Group 1. sperm separation (SS): Percoll (Sigma, St. Louis, MO, USA), fertilization medium (FM): TALP-Fert (TFM), embryo culture media (ECM): G1/G2 (version 3, Vitrolife, Englewood, CO, USA). Group 2. SS: Percoll, FM: Bovine vitro Fert (Cook, Brisbane, Australia), ECM: Bovine vitro Blast/Bovine vitro Cleave (Cook); and Group 3. SS: EquiPure (Nidacon, Spectrum Technologies, Healdsburg, CA, USA), FM: TFM, ECM: G1/G2. Oocytes were obtained from slaughterhouse ovaries and matured in vitro (Looney et al. 1994 Theriogenology 41, 67). IVF was conducted using frozen/thawed semen from one bull. Semen was separated by centrifugation at 700g for 30 min in the given density gradients; Percoll was used in a 45% to 90% gradient. Sperm viability after separation was assessed by fast-green/eosin stain (Sigma). IVF was carried out in 0.5 mL of the given fertilization medium supplemented with PHE1 and heparin (10 μg/mL), in humidified 5% CO2 in air atmosphere at 38.7°C. Final sperm concentration in the IVF wells was 1 × 106/mL. In Experiment 1, a total of 368 oocytes (2 replicates) were fixed and stained (Hoechst 33342, Sigma) 24 h post-IVF to assess sperm penetration (Group 1, n = 128, Group 2, n = 108, Group 3, n = 132). In Experiment 2, a total of 400 embryos (2 replicates) were cultured in 0.5 mL of the given culture medium under mineral oil in a 5% O2, 5% CO2, 90% N2 atmosphere at 38.7°C with high humidity for 112 h before fixation and staining. Embryos in Groups 1 (n = 129) and 3 (n = 139) and Group 2 (n = 132) were changed to G2 and Cleave media, respectively, at 84 h. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 218 vs. 383 × 106 for EquiPure; P < 0.05), but resulted in higher total motility (60% vs. 41%, respectively; P < 0.05) and higher viability (93% vs. 70%, respectively; P < 0.05) of separated sperm. In Experiment 1, rates of normal fertilization were significantly lower for Group 3 (58%) than for Groups 1 and 2 (74% and 77%, respectively, P < 0.05). In Experiment 2, rates of development to <8, 9 to 16, and >16 cells at 112 h were not significantly different among groups (43, 48, and 46% for Group 1; 22, 18, and 31% for Group 2; and 35, 34, and 23% for Group 3, respectively; P > 0.1). These results indicate that the commercial separation medium, EquiPure, may be associated with lowered sperm motility, viability, and fertilization rates when compared to a standard medium (Percoll) for bovine sperm separation. Commercial fertilization and embryo culture media (Bovine vitro Fert, Cleave, and Blast) provided equivalent embryo development to that currently in use by our laboratory (TFM, G1/G2).

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