Biomedical subjects

G E Seidel

Publications and source records attributed to G E Seidel.

At least 19 recordsLinked to original sources

PubMed2006

Effect of macromolecules in solutions for vitrification of mature bovine oocytes.

This study was designed to evaluate vitrification procedures for in vitro matured bovine oocytes for efficient blastocyst production after warming, IVF and culture. A second goal was to replace serum as the macromolecular component of the vitrification solution, without compromising efficacy. The first experiment compared two containers, open pulled straws (OPS) versus cryoloops, and two vitrification protocols: short equilibration (H-TCM-199+10% EG+10% DMSO+20% FCS for 30s, followed by H-TCM-199+20% EG+20% DMSO+20% FCS+0.48M galactose for 20s) versus long equilibration (H-TCM-199+3% EG+20% FCS for 10min, followed by H-TCM-199+31% EG+20% FCS+1M galactose for 20s). Subsequent experiments used only cryoloops and the short equilibration protocol to evaluate the effect of replacing FCS with defined macromolecules (BSA, Ficoll, PVP, and PVA) in vitrification solutions. Cryoloops were superior to OPS for vitrification of oocytes as determined by blastocyst production (P<0.05). The short and long vitrification protocols gave similar results. The presence of macromolecules in vitrification solutions for bovine oocytes was necessary for acceptable post-warming developmental capacity; 20% FCS, 1% and 2% BSA, 6% and 18% Ficoll, 6% and 20% PVP, 1% PVA, and the combinations of 18% Ficoll+1% BSA, and 6% PVP+1% BSA provided similar protection during vitrification of oocytes; development ranged from 14.8% to 23.0% blastocysts/oocyte, which was not different (P>0.05) from non-vitrified controls (26.9-34.0% blastocysts/oocyte). Too much (6%) and too little (0.3%) BSA, and 0.3% PVA for vitrification resulted in lower blastocyst production (P<0.05) relative to unvitrified oocytes.

Animals↗

PubMed2006

Vitrification of in vitro-produced bovine embryos by addition of ethylene glycol in one-step.

The objective of this study was to simplify two-step addition of cryoprotectant for vitrification of bovine embryos by developing a one-step procedure. Survival was calculated as a percentage of non-vitrified controls developed from the same batch of oocytes. In experiment 1, bovine blastocysts were vitrified following one- or two-step addition of cryoprotectant. Exposure of embryos to cryoprotectant in one-step resulted in survival rates not significantly lower (p > 0.1) than those obtained by two-step addition (85% vs 98%, respectively). Based on these results, experiments 2-4 were designed to test one-step addition of cryoprotectant more rigorously. Experiment 2 exposed day 7 blastocysts to 6, 7 or 8 M ethylene glycol for 2.5 or 3.5 min. At 24 h post-vitrification, survival of embryos was similar, irrespective of ethylene glycol concentration or exposure time (6 M 38%, 7 M 51%, 8 M 59%; 2.5 min 54%, 3.5 min 45%). In experiment 3, blastocysts were exposed to 7 M ethylene glycol for shorter times (30 or 60 s); 30 s exposure resulted in decreased survival (8% vs 31%, p < 0.05). Experiment 4 concerned one-step addition of cryoprotectant to day 6 bovine morulae, exposed to 7 M ethylene glycol for 1 or 1.5 min. There was no difference in survival between exposure times of 1 or 1.5 min (28% vs 45%, respectively; p > 0.1). It is unclear why many embryos survive vitrification with one-step addition of cryoprotectant, but others do not. Although, one-step addition of cryoprotectant simplifies the vitrification procedure, survival rates were inadequate for routine cryopreservation of in vitro-produced bovine embryos.

Animals↗

PubMed2005

In vitro assessment of a direct transfer vitrification procedure for bovine embryos.

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (P<0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 degrees C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 degrees C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 degrees C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.

Animals↗

PubMed2005

Embryo production from superovulated cattle following insemination of sexed sperm.

Two trials were conducted to ascertain fertilization rate, embryo quality and numbers of transferable embryos in superovulated heifers and cows inseminated with sexed sperm. Inseminates contained 2 x 10(6), 10 x 10(6) or 20 x 10(6) total sperm enriched for the X- or Y-chromosome ( approximately 90%) by flow cytometry/cell sorting. Non-sexed inseminates contained 40 x 10(6) total sperm. Donors in each trial were allocated to one of each of the bulls included in that study. Each donor was inseminated with frozen/thawed sperm from the same bull for each treatment in successive courses of superstimulation with twice daily i.m. injections of FSH for 4 d. Heifers and cows were inseminated 12 and 24 h after visually observed standing estrus in Trial 1. In Trial 2, a single timed inseminate was used 70-72 h following PGF(2alpha). Ova/embryos were collected non-surgically 7-7.5 d after insemination. In both trials, fewer ova were fertilized with sexed versus non-sexed treatments and with 2 x 10(6) sexed sperm compared to higher doses (P < 0.05). However, insemination of 20 x 10(6) total sexed sperm of >or=90% purity resulted in similar numbers of transferable embryos of the desired sex compared to that for non-sexed sperm.

Animal Husbandry↗

PubMed2005

Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos.

Experiments were conducted to determine viability of equine embryos in vivo after vitrification. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G); the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Embryos were warmed in a two-step dilution and transferred into uteri of recipients. No pregnancies were observed after transfer of blastocysts >300 microm (n = 3). Transfer of morulae or blastocysts < or = 300 microm resulted in four embryonic vesicles (4/6, 67%). In a second experiment, embryo recovery per ovulation was similar for collections on Day 6(28/36, 78%) versus Days 7 and 8(30/48, 62%). Embryos < or = 300 and >300 microm were vitrified, thawed and transferred as in Experiment 1. Some embryos < or = 300 microm were also transferred using a direct-transfer procedure (DT). Embryo development rates to Day 16 were not different for embryos < or = 300 microm that were treated as in Experiment 1(10/22, 46%) or transferred by DT (16/26, 62%). Embryos > 300 microm (n = 19) did not produce embryonic vesicles.

Animals↗

PubMed2005

High pressure flow cytometric sorting damages sperm.

Sexing sperm by high-speed flow cytometry subjects them to high pressure. The routine operating pressure of the MoFlo SX flow cytometer for sperm sorting for commercial production has been 50 pounds/square inch (psi), with a standard 70 microm standard nozzle tip. It was hypothesized that lowering the sorting pressure could reduce sperm damage. Therefore, a series of experiments using semen from six bulls, sorted with three MoFlo SX sorters, was conducted to determine optimal pressure. An additional experiment was done with stallion spermatozoa. In Experiment 1, sorting at 30 psi compared to 50 psi with the 70 microm nozzle tip increased sperm motility post-thaw at 30 min and 2h from 40.5 to 48.0% and 30.0 to 40.2%, respectively (P<0.05). In Experiment 2, 49, 43, 37, 31, and 25 psi resulted in 24.2, 32.8, 35.6, 37.5, and 39.8% progressively motile spermatozoa post-thaw (P<0.05). In Experiment 3, 3 pressures (50, 40, 30 psi)x2 sorting methods were further evaluated. At 50, 40, and 30 psi, respective mean sperm motilities at 30 min were 44.8, 48.6, and 49.6% (P<0.05), and percentage of live spermatozoa were 51.7, 55.7, and 57.8% (P<0.05). The improvement of post-sort sperm quality with lowered pressure was also evident in stallion spermatozoa. After sorting at 30, 40 and 50 psi were 40.6, 34.5 and 30.1% motile spermatozoa (P<0.1), and were 76.7, 72.5 and 67.8% (P<0.05) live spermatozoa (determined by SYBR-14/propidium iodide staining). In Experiment 4 sorter performance was evaluated with two pressures (40 and 50 psi)x2 staining concentrations of bovine spermatozoa (75 x 10(6) and 100 x 10(6)mL(-1)). Lowering pressure to 40 psi did not lower sort rate and purity when compared to 50 psi (P>0.05), and higher sperm concentration during staining increased sort rate (P<0.05). In conclusion, lowering pressure of the MoFlo SX flow cytometer for sperm sorting from 50 psi (standard pressure) to 40 psi clearly improved sperm quality without a significant decrease in sorter performance.

Animals↗

PubMed2005

Hysteroscopic or rectally guided, deep-uterine insemination of mares with spermatozoa stored 18 h at either 5 degrees C or 15 degrees C prior to flow-cytometric sorting.

Practical application of sex-selected spermatozoa in the horse industry would be greatly improved by the ability to develop simplified methods for shipping, storing, and inseminating sex-selected spermatozoa. Acceptable pregnancy rates have been achieved using fresh sex-sorted stallion sperm, however many stallion owners are reluctant to send their stallions to the sorter location for collection during the breeding season. Furthermore, the technology would be more applicable if the hysteroscopic insemination technique was not necessary for adequate pregnancy rates. Hysteroscopic insemination requires expensive equipment and specially trained personnel. In the present study, stallion sperm were sex-sorted after being stored at either 5 degrees C or 15 degrees C for 18 h. Twenty million sex-sorted sperm were then inseminated using one of two insemination techniques: the hysteroscopic method or the rectally guided, deep-uterine technique. Results were determined based on 16-day pregnancy status. A first-cycle pregnancy rate of 72% (18/25) was achieved when sperm were shipped at 15 degrees C, sex-sorted, and then inseminated using the hysteroscopic method. With these results, it can be concluded that stallions are not necessary at the sorter location to achieve acceptable fertility with sex-sorted sperm. There was a tendency for more mares to become pregnant when sperm were shipped at 15 degrees C prior to sorting, when compared to shipment at 5 degrees C. Similarly, there was a tendency for more mares to become pregnant when hysteroscopic insemination was utilized, when compared to the rectally guided, deep-uterine technique. These trends suggest that if larger group numbers were available, significant differences between the treatments may be revealed.

Animals↗

PubMed2004

Methanol as a cryoprotectant for equine embryos.

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.

Animals↗

PubMed2004

Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm.

The objective was to determine the optimal concentration of heparin for sperm capacitation, as well as the optimal sperm concentration for in vitro fertilization using flow cytometrically-sorted sperm from individual bulls. A total of 5327 bovine oocytes and sperm from four bulls were examined. Oocytes from slaughterhouse ovaries were matured in TCM199 for 22-24 h. Flow cytometrically-sorted sperm as well as unsorted control sperm from the same bulls were cryopreserved. For sperm from each of the four bulls, oocytes were inseminated in a three-by-three factorial design plus one control group (three heparin concentrations: 0, 2, and 10 microg/ml and three sperm concentrations: 0.5 x 10(6), 1.5 x 10(6), and 4.5 x 10(6) ml(-1); 10 microg/ml of heparin and 1.5 x 10(6) ml(-1) of sperm were used for the unsorted control). Presumptive zygotes were cultured in chemically defined media, CDM-1 and CDM-2 for 52-54 h and 96 h, respectively. Samples of about 10 oocytes from each of the 10 treatment groups per replicate were fixed at 18-20 h after insemination to determine sperm pronuclei formation and polyspermy. Increased polyspermy resulted as heparin and sperm concentrations increased (P < 0.05). A higher rate of polyspermy was found in oocytes inseminated with unsorted control sperm compared with sorted sperm (P < 0.05). Sperm of one of four bulls tested required no heparin and lower concentration (0.5 x 10(6) ml(-1)) to obtain optimal cleavage and blastocyst rates while optimal parameters for another bull were higher heparin (10 microg/ml) and sperm concentrations (4.5 x 10(6) ml(-1)). Optimal parameters for the other two were intermediate levels of heparin and sperm. Sperm appeared to be partially capacitated during the flow cytometric-sorting process used for sex pre-determination. When heparin and sperm concentrations were optimized for individual bulls, blastocyst production per oocyte was similar for sorted and unsorted sperm for three of the four bulls studied.

Animals↗

PubMed2004

Improved cleavage of bovine ICSI ova cultured in heparin-containing medium.

Although heparin plays an important role in bovine sperm capacitation, there is no direct evidence for a role in embryonic development. The present study was designed to examine the effect of heparin on early development of bovine zygotes obtained by intracytoplasmic sperm injection (ICSI). Spermatozoa were treated with or without heparin, and the ICSI ova were cultured in a chemically defined medium + BSA, with or without heparin. Treatment of spermatozoa with heparin before ICSI or heparin in post-ICSI culture medium for 18 or 24h had a beneficial effect on pronuclear formation, cleavage rate (63% versus 76-83%), and number of cells in blastocysts (68 cells versus 82-109 cells; P < 0.05).

Animals↗

PubMed2004

Oocyte transfer in mares with intrauterine or intraoviductal insemination using fresh, cooled, and frozen stallion semen.

The objectives were to compare embryo development rates after oocyte transfer with: (1) intrauterine or intraoviductal inseminations of fresh semen versus intraoviductal insemination of frozen semen; (2) intraoviductal versus intrauterine inseminations of cooled semen. In Experiment I, oocytes were transferred into the oviduct, and recipients were inseminated into the uterus with 1 x 10(9) fresh spermatozoa, or into the oviduct with 2 x 10(5) fresh or frozen-thawed spermatozoa. In Experiment II, semen was cooled to 5 degrees C before intrauterine insemination with 2 x 10(9) spermatozoa or intraoviductal inseminations of 2 x 10(5) spermatozoa (deposited with the oocytes). In Experiment I, embryo development rates were similar (P>0.05) for intrauterine versus intraoviductal inseminations when fresh semen was used (8/14, 57% and 9/11, 82%, respectively). However, embryo development rates were lower (P<0.05) when frozen spermatozoa were placed within the oviduct (1/12, 8%). In Experiment II, embryo development rates were higher (P<0.05) when cooled semen was used for intrauterine (19/23, 83%) versus intraoviductal (4/16, 25%) inseminations. We concluded that intraoviductal insemination can be successfully performed using fresh spermatozoa. However, the use of cooled and frozen spermatozoa for intraoviductal inseminations was less successful, and needs further investigation.

Animals↗

PubMed2004

Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting.

The objectives of this study were to determine whether calves produced by sexed sperm differed from controls and to what extent the sex ratio of calves was altered by the sexing procedure. Data were collected from 1,169 calves produced from sperm sexed by flow cytometry/cell sorting after staining with Hoechst 33342, and 793 calves produced from control sperm during breeding trials between 1997 and 2001. Least squares ANOVA were completed using factors of treatment (sexed vs. control sperm), 19 management groups from 13 field trials, and calf sex. Responses analyzed include gestation length, birth weight, calving ease, calf vigor, weaning weight, abortion rate, and death rates (neonatal and through weaning). No significant difference was observed for any response due to treatment or treatment interactions (P > 0.10). Therefore, calves produced from sexed sperm grew and developed normally both pre- and postnatally. A neurological disorder was observed in four control calves and one sexed calf from one farm. No gross anatomical abnormalities were reported for any calves in the study. Differences were observed for all responses among management groups (P < 0.03 for abortions and P < 0.01 for all other responses). Heifer and bull calves differed (P < 0.001) in gestation length (278.4 and 279.6 d), birth weight (32.8 and 35.2 kg), calving ease (1.15 and 1.30), and weaning weight (233 and 247 kg). Gestation length did not affect characteristics of calves. The sex ratio at birth of calves from unsexed control sperm was 49.2% male. Sexing accuracy of X-sorted sperm was 87.8% female calves, and Y-sorted sperm produced 92.1% male calves. Flow cytometry/cell sorting can be used to preselect sex of calves safely with approximately 90% accuracy.

Analysis of Variance↗

PubMed2003

Economics of selecting for sex: the most important genetic trait.

Over 20,000 calves have resulted from artificial insemination (AI) of cattle with sexed, frozen/thawed sperm in the course of experimentation in several countries, and from commercial sales in the United Kingdom. This technology likely will become commercially available in many countries within a few years. Accuracy of the process is about 90% for either sex, and resulting calves appear to be no different from non-sexed controls in birthweight, mortality, rate of gain, and incidence of abnormalities. The main determinants of the extent of use of sexed sperm will be pregnancy rate and cost. Fertility of low doses (1.5 x 10(6)-2 x 10(6)) of sexed, frozen sperm for AI of heifers usually has been in the range of 70-80% of unsexed sperm at normal doses (10 x 10(6)-20 x 10(6) sperm) in well managed herds; it has been lower in poorly managed herds, and likely will be lower with lactating dairy cows. It is expected that fertility of sexed sperm will increase significantly due to very recent improvements in the hydrodynamics of the sexing process and potential improvements in cryopreservation procedures. It is unclear how sexed sperm will be priced; the cost of sexed sperm for cattle will likely be more than double the cost of unsexed sperm in most markets. The economic benefit of using sexed sperm also will depend on the baseline fertility of the herd since at lower fertility, it takes more doses of semen per calf produced. It is noted that for a small percentage of elite cattle, the economics of using sexed sperm do not depend primarily on increased production or efficiency of producing meat or milk, but rather on factors such as scarcity, tradition, cattle show winnings, and biosecurity during herd expansion. Until sorting efficiencies improve and costs decline, sales likely will be limited primarily to these niche markets. With near normal fertility and a premium for sexing in the range of US$ 10 per insemination dose, sexed sperm likely would become economically and environmentally beneficial for many, if not most populations of cattle being bred by AI.

Animals↗

PubMed2003

Intracytoplasmic sperm injection of bovine oocytes with stallion spermatozoa.

Five experiments were designed to study the fertilizability and development of bovine oocytes fertilized by intracytoplasmic sperm injection (ICSI) with stallion spermatozoa. Experiment 1 determined the time required for pronuclear formation after ICSI. Equine sperm head decondensation began 3 h after ICSI; 42% were decondensed 6 h after ICSI. Male pronuclei (MPN) began to form 12 h after ICSI. Female pronuclei (FPN), however, formed as early as 6 h after ICSI. In Experiment 2, ionomycin, ionomycin plus 6-dimethylaminopurine (DMAP), and thimerosal were used to activate ICSI ova. None of the ICSI ova cleaved after treatment with thimerosal. Ionomycin activation after 24 and 30 h of oocyte maturation resulted in 29 and 48% cleavage rates, respectively. Ionomycin combined with DMAP resulted in 49, 6 and 3% cleavage, morula and blastocyst rates, respectively, when oocytes were activated after 24 h maturation. In Experiment 3, rates of cleavage (45-60%) and development to morulae (4-13%) and blastocysts (1-5%) stages following ICSI were not different (P>0.05) among three stallions. Treatment of stallion spermatozoa with ionomycin did not affect cleavage or development of ova fertilized by ICSI. The chromosomal constitution of blastocysts derived from ICSI was bovine, not bovine and equine hybrids. In Experiment 4, to make male and FPN form synchronously, colchicine and DMAP were used for 4 h to inhibit oocytes at metaphase during activation; 63% of oocytes were still at metaphase 8h after ICSI when treated with colchicine, and 50% of sperm nuclei were decondensed. About 18 h after ICSI, 21 and 50% male and FPN had formed, respectively, but cleavage rates were low, and only 1% developed to morulae. In Experiment 5, to test if capacitated equine sperm could fuse with the bovine oolemma, capacitated spermatozoa were injected subzonally (SUZI). Of the 182 SUZI oocytes, 49 (27%) contained extruded second polar bodies. After activation of oocytes with second polar bodies, 44, 22 and 15% developed to 2-, 4- and 8-cell stages, respectively, but development stopped at the 8-cell stage. None of the unactivated oocytes cleaved. In conclusion, equine spermatozoa can decondense and form MPN in bovine oocytes after ICSI, but subsequent embryonic development is parthenogenetic with only bovine chromosomes being found.

Adenine↗

PubMed2003

Effect of heparin on cleavage rates and embryo production with four bovine sperm preparation protocols.

Heparin is often added to fertilization media to induce sperm capacitation. However, recent observations from the bovine IVF industry have indicated that heparin may not be necessary to induce sperm capacitation when cryopreserved bovine sperm are separated through Percoll gradients. The objective of these studies was to determine if the addition of heparin to fertilization media was required following separation of frozen-thawed bovine sperm. Experiment 1 was conducted to determine the effect of heparin on cleavage rates and embryo production when using Percoll, BSA or washing sperm separation protocols. Experiment 2 was conducted to determine the effect of heparin on cleavage rates and embryonic development when using a new sperm separation protocol (PureSperm). In Experiment 1, regardless of sperm separation protocol, heparin increased cleavage rates and embryo production (P<0.05). Further, cleavage rates and embryo production were higher for the Percoll procedure compared to either BSA or washing sperm separation protocols (P<0.05). In Experiment 2, there was no difference between Percoll and PureSperm separation protocols on cleavage rates or embryo production (P>0.05). However, heparin increased cleavage rates for both protocols and improved embryo production for the PureSperm protocol (P<0.05). In conclusion, use of heparin in fertilization media improves in vitro embryo production when using cryopreserved bovine sperm. Furthermore, PureSperm can be used as an alternative to Percoll for bovine sperm separation.

Animals↗

PubMed2003

Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically sorted, stained and unsorted sperm from different bulls.

The objective of this study was to determine the effect of staining bovine sperm, with or without flow cytometry, on in vitro fertilization of bovine oocytes and blastocyst development. Bovine oocytes (n=4273) were fertilized with frozen-thawed sperm from three bulls that was: stained with Hoechst 33342 and sorted (into X- or Y-chromosome-bearing sperm) with flow cytometry; stained but not sorted; and not stained or sorted (Control). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 (supplemented with 10% fetal calf serum and 15 ng FSH, 1.0 microg LH, 1.0 microg E2/ml) for 22-24h at 39 degrees C in 5% CO(2) in air with maximum humidity. Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1) 6-7h after insemination and cultured for 65-66h. Embryos that had cleaved by 72h post-insemination were cultured an additional 96h in CDM-2 containing 0.12 IU insulin/ml. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7-8 after insemination, respectively. There was no significant difference in blastocyst rate among the three types of sperm; however, cleavage rates with stained and sorted sperm (53.1%) and unsorted, stained sperm (59.9%) were lower (P<0.05) than Control sperm (69.7%). Furthermore, there were significant differences due to semen from different bulls in cleavage and blastocyst rates.

Animals↗

PubMed2003

Fertility assessment through heterospermic insemination of flow-sorted sperm in cattle.

The ability to assess fertility of bovine sperm accurately and rapidly would be very useful for research and applications to the cattle industry. Sperm motility and other in vitro tests of sperm normality are only partially correlated with fertility, and lengthy breeding trials are expensive and time consuming. Heterospermic insemination by mixing sperm from more than one male provides an in vivo method to assess relative fertility among bulls that can be economical and rapid. Sperm that had been flow-sorted and cryopreserved from four groups of four bulls were inseminated in all combinations of three bulls within groups into nonsuperovulated heifers or superovulated heifers. Embryos were collected nonsurgically between d 13.5 and 20 following estrus and evaluated for paternity by genotyping. Following determination of paternity, a heterospermic index was created for each bull using a maximum likelihood function. These indices ranged from 0.22 +/- 0.15 to 2.43 +/- 0.43 (mean = 1.00, with a higher value indicative of greater fertility). In all four groups, either the high- or low-fertility bull was identified (P < 0.05) using a total of 25 to 36 genotypable embryos from nonsuperovulated heifers. The heterospermic rankings of bulls were similar for single and superovulated heifers for one group of bulls, but dissimilar for a second group. Heterospermic insemination followed by genotyping of embryos proved to be efficacious for rapidly ranking fertility of flow-sorted sperm from bulls when females were not superovulated, but results were less clear when females were superovulated.

Animals↗

PubMed2003

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes.

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm.

8-Bromo Cyclic Adenosine Monophosphate↗