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Biomedical subjects

J J Rutledge

Publications and source records attributed to J J Rutledge.

At least 19 recordsLinked to original sources
PubMed2005

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle.

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one (n = 31 recipients) or two (n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127=17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo (P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 microg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.

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PubMed2005

In vitro production of bovine embryos using sex-sorted sperm.

The objective of this study was to investigate the suitability of sex-sorted sperm for producing viable in vitro embryos for subsequent transfer into recipient cows and heifers on commercial dairy farms. From August 2002 to June 2003, ovaries were collected from 104 producer-nominated Holstein donor cows on seven Wisconsin farms via colpotomy or at slaughter. Oocytes (N=3526) were aspirated from these ovaries, fertilized 22+/-0.2h later, and cultured to the morula or blastocyst stage. The fluorescence-activated cell sorting ("Beltsville") approach was used to produce (primarily) X-bearing sperm from the ejaculates of three young Holstein sires, and 365 transferable embryos were produced. On average, 3.6+/-0.3 (means+/-S.E.M.) transferable embryos were produced per donor, including 1.4+/-0.2 (Grade 1), 1.5+/-0.2 (Grade 2), and 0.7+/-0.1 (Grade 3) embryos. Number of usable oocytes per donor (33.9+/-3.3) and percent cleavage (51.1+/-1.9) were significant predictors of the number of blastocysts that developed. Mean conception rates for the resulting in vitro embryos were 34.2+/-1.6% in yearling heifer recipients and 18.2+/-0.7% in lactating cow recipients. Additional oocytes (N=3312) from ovaries of anonymous donors (N unknown) collected at a commercial abattoir were fertilized using unsorted sperm, and the percentage of these that developed to blastocyst stage (20.1+/-2.9) was greater (P<0.05) than the corresponding percentage (12.2+/-2.3) achieved with sex-sorted sperm using oocytes (N=1577) from the same source. In summary, we inferred that in vitro embryo production may be a promising application of sex-sorted sperm in dairy cattle breeding, but that the biological causes of impaired embryo development in vitro and compromised conception rates of transferred embryos should be further investigated.

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PubMed2005

Parturition following transfer of embryos produced in two media under two oxygen concentrations.

In vitro-produced blastocysts were transferred singly to 99 Angus crossbred recipients. Culture treatments were a 2 x 2 factorial of medium (KSOM or SOF) and oxygen concentration (5 or 20%). At parturition, birth weight and frame measurement before colostrum intake were recorded. Fetal membranes were collected; distribution and diameter of cotyledons was recorded. Cotyledon surface area was calculated. Culture with 5% O2 tended to yield smaller birth weights than culture with 20% O2 (39.7+/-1.3 kg versus 43.1+/-1.4 kg; P < 0.1); this effect was pronounced in KSOM, suggesting a medium by oxygen interaction (P < 0.1). When expressed on a body weight basis, calves born following culture with 20% O2 had consistently smaller skeletal measurements than those from culture with 5% O2. Culture with 20% O2 significantly increased individual cotyledon areas both overall and in the fetal horn and cotyledon surface area in the fetal horn. Overall, individual cotyledons were 32% larger when culture involved 20% O2 versus 5% O2; in the fetal horn the increase was 49%. Cotyledon surface area was greater for 20% compared to 5% O2 culture, though a medium by oxygen interaction was also significant (P < 0.05). Cotyledon surface area in the nonfetal horn was greater for KSOM fetal membranes than those from culture in SOF. There was a significant medium by oxygen interaction for total cotyledon number. These data demonstrate culture system-specific effects on calf and fetal membrane traits.

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PubMed2005

In vitro production of Holstein embryos using sex-sorted sperm and oocytes from selected cull cows.

The objective of this study was to explore potential synergies between sex-sorted sperm and in vitro embryo production for generating replacement heifers on commercial dairy farms. Selected involuntary cull cows (i.e., genetically suitable cows that were culled due to injury, illness, or infertility) from 7 Wisconsin farms were used as donors, and ovaries were collected via colpotomy or at the time of slaughter. Oocytes were aspirated, fertilized in vitro with sex-sorted sperm 22 +/- 0.2 h later, cultured, matured for 7 to 8 d, and transferred into recipient cows and heifers on the farms from which the cull cows originated. From August 2002 to June 2003, ovaries were recovered from 104 Holstein donors. Sex-sorted sperm from 3 Holstein sires (obtained via fluorescence-activated cell sorting) were used. A total of 365 transferable embryos were produced, an average of 3.6 +/- 0.3 per donor. However, due to limited availability of recipient animals, only 272 (fresh) embryos were transferred, an average of 2.6 +/- 0.3 per donor. A random subset of recipients received an injection (i.m.) of GnRH (100 microg) at the time of embryo transfer. When lactating cows were used as recipients, mean conception rates were 16.3% for recipients identified based on standing estrus and 20.0% for recipients synchronized using a timed breeding program (Ovsynch). Conception rates for in vitro-produced embryos were lower than corresponding conception rates for control cows inseminated using unsorted semen. When virgin heifers were used as embryo recipients (all standing estrus), the mean conception rate was 34.2%. The following effects significantly impacted conception rate: farm, season, recipient group (cow vs. heifer), sire of embryo, and GnRH injection. Of 40 full-term calves generated using sex-sorted semen, 37 were female. These results suggest that "low-cost" in vitro embryo production using cull cows as donors, in conjunction with sex-sorted sperm, could be an effective tool in dairy cattle breeding programs, but only if conception rates can be improved.

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PubMed2004

Early mammalian embryo development depends on cumulus removal technique.

Cumulus removal (CR) at the zygote stage is necessary for most mammalian in vitro production (IVP). Present techniques use high fluidic stresses (vortexing) or mechanical stress with enzymatic treatment (pipetting) to remove cumulus. Herein a recently developed microfluidic device for cumulus removal from zygotes is compared with traditional vortexing. Microfluidic CR (microFCR) increased development on day 2 (20 +/- 4% to 35 +/- 6%, p < 0.01) and blastocyst formation at day 8 (33 +/- 1% to 57 +/- 5%, p < 0.01) when compared to vortex CR. Vortexing effects on embryo development were studied; 15, 30 and 120 s vortex doses. Development at day 2 was inversely proportional to duration of vortexing. An in situ transcription assay was used to assess biochemical activity of zygotes after cumulus removal. There was a spike of RNA transcription of vortexed zygotes at 2 h post CR not seen in the microfluidic treatment. These results suggest the potential for microfluidic methods to enhance production efficiencies while providing insight into basic developmental mechanisms.

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PubMed2004

Embryonic disc development and subsequent viability of cattle embryos following culture in two media under two oxygen concentrations.

Bovine embryos were produced in vitro using a 2 x 2 design of modified medium (KSOM or SOF) and oxygen concentration (5% or 20%). Day 7 blastocysts were transferred in bulk (n = 11, on average) to recipient heifers and recovered non-surgically at Day 14. In two replications of a Latin square, eight heifers received embryos from each combination of factors. Recovered embryos were evaluated for trophoblast length and width, as well as the presence and diameter of an embryonic disc (ED). An ED was detected in a higher percentage of embryos that had been cultured in KSOM than SOF (72% v. 46%, respectively; P < 0.05). The aim of a second series of experiments was to associate Day 14 morphology with subsequent developmental capacity. In vitro-produced blastocysts were transferred (n = 17-20) on Day 7 to each of eight heifers and recovered at Day 14. Thirty-eight blastocysts were retransferred to heifers following morphological evaluation. Embryos in which an ED with no signs of degeneration had been detected maintained more pregnancies than other embryos in which an ED had either shown signs of degeneration or had not been detected (5/8 v. 2/30, respectively; P < 0.01). Further investigation into ED integrity at the elongating stage may contribute to our understanding of pregnancy establishment and maintenance.

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PubMed2003

Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation.

Developmental competence of mammalian oocytes is compromised by currently available oocyte cryopreservation protocols. Experiments were designed to examine the effect of three cryopreservation protocols on the integrity of bovine oocyte DNA. In vitro matured bovine oocytes were cryopreserved either by slow cooling, vitrification in 0.25 ml straws, or in open pulled straws. After thawing/warming, recovered oocytes were immediately subjected to morphological evaluation. Morphologically intact oocytes underwent comet assay to detect cryoinjury at DNA level. All cryopreservation protocols resulted in significant morphological damage as well as DNA damage compared to unfrozen control. Among the morphologically intact oocytes, there was no difference among protocols in the number of oocytes displaying DNA damage. However, oocytes that had been cryopreserved by slow cooling or by vitrification in open pulled straws exhibited more damage than those vitrified in 0.25 ml straws in the extent of DNA damage. If we combine the number of oocytes with morphological damage and oocytes with DNA damage, oocytes cooled by slow cooling resulted in the most damage. This experiment demonstrated that oocyte DNA is a target of cryoinjury and different protocols result in different degrees of damage.

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PubMed2003

Use of insulin-like growth factor-I during embryo culture and treatment of recipients with gonadotropin-releasing hormone to increase pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed, lactating cows.

An experiment was conducted to determine whether pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed cows could be improved by 1) culturing embryos in the presence of IGF-I and 2) treating recipients with GnRH. Lactating Holstein cows (n = 260) were synchronized using a timed ovulation protocol. Embryos were produced in vitro and cultured with or without 100 ng/mL of IGF-I. On d 7 after anticipated ovulation (d 0), a single embryo was transferred to all recipients with a palpable corpus luteum (n = 210). A subset of recipients (n = 164) was injected with either GnRH or placebo on d 11. Plasma progesterone concentrations on d 0 and 7 were used to determine the synchrony of recipients. Pregnancy was diagnosed at d 53 and 81 by rectal palpation. Among all recipients, transfer of IGF-I-treated embryos increased pregnancy rate at d 53 (P < 0.05) and tended to increase pregnancy rate at d 81 (P < 0.06). Calving rate also tended to be higher for recipients that received IGF-I-treated embryos (P < 0.07). Among the subset of synchronized recipients (n = 190), pregnancy rate at d 53 and d 81 and calving rate were higher (P < 0.05) for IGF-I-treated embryos. The GnRH tended to increase pregnancy rate at d 53 for all recipients (P < 0.08) and the subset of synchronized recipients (P < 0.10). There were no effects of GnRH (P > 0.10) for pregnancy rate at d 81 and calving rate. The overall proportion of male calves was 64.3%. There was no effect (P > 0.10) of embryo treatment or GnRH on the birth weight or sex ratio of calves. Results of this experiment indicate that treatment of embryos with IGF-I can improve pregnancy and calving rates following transfer of in vitro-produced embryos. Further research is necessary to determine whether the treatment of recipients with GnRH is a practical approach to increase pregnancy rates following in vitro embryo transfer.

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PubMed2002

Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions.

Timed embryo transfer (TET) using in vitro produced (IVP) embryos without estrus detection can be used to reduce adverse effects of heat stress on fertility. One limitation is the poor survival of IVP embryos after cryopreservation. Objectives of this study were to confirm beneficial effects of TET on pregnancy rate during heat stress as compared to timed artificial insemination (TAI), and to determine if cryopreservation by vitrification could improve survival of IVP embryos transferred to dairy cattle under heat stress conditions. For vitrified embryos (TET-V), a three-step pre-equilibration procedure was used to vitrify excellent and good quality Day 7 IVP Holstein blastocysts. For fresh IVP embryos (TET-F), Holstein oocytes were matured and fertilized; resultant embryos were cultured in modified KSOM for 7 days using the same method as for production of vitrified embryos. Excellent and good quality blastocysts on Day 7 were transported to the cooperating dairy in a portable incubator. Nonpregnant, lactating Holsteins (n = 155) were treated with GnRH (100 microg, i.m., Day 0), followed 7 days later by prostaglandin F2alpha (PGF2alpha, 25 mg, i.m.) and GnRH (100 microg) on Day 9. Cows in the TAI treatment (n = 68) were inseminated the next day (Day 10) with semen from a single bull that also was used to produce embryos. Cows in the other treatments (n = 33 for TET-F; n = 54 for TET-V) received an embryo on Day 17 (i.e. Day 7 after anticipated ovulation and Day 8 after second GnRH treatment). The proportion of cows that responded to synchronization based on plasma progesterone concentrations on Day 10 and Day 17 was 67.7%. Pregnancy rate for all cows on Day 45 was higher (P < 0.05) in the TET-F treatment than for the TAI and TET-V treatments (19.0 +/- 5.0,6.2 +/- 3.6, and 6.5 +/- 4.1%). For cows responding to synchronization, pregnancy rate was also higher (P < 0.05) for TET-F than for other treatments (26.7 +/- 6.4, 5.0 +/- 4.3, and 7.4 +/- 4.7%). In the TET-F treatment group, cows producing more milk had lower (P < 0.05) pregnancy rates than cows producing less milk. In conclusion, ET of fresh IVP embryos can improve pregnancy rate under heat stress conditions, but pregnancy rate following transfer of vitrified embryos was no better than that following TAI.

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PubMed2001

Use of embryo transfer and IVF to bypass effects of heat stress.

Although heat stress has multiple effects to lower pregnancy rate in lactating dairy cows, a major pathway is in its effects on the early cleavage stage embryo. Conceptually, and in practice, higher pregnancy rates can be obtained with transfer of late cleavage stage embryos. The literature is reviewed, and conclusion is made that application of these technologies may be in part, a solution to this long-standing problem.

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PubMed2000

Estimates of heterosis for in vitro embryo production using reciprocal crosses in cattle.

In vitro embryo production and exploitation of heterosis are two methods of increasing productivity and accelerating genetic progress in many cattle production systems. However, it is not known if heterosis exists in bovine embryos produced in vitro. Tests for heterosis in in vitro embryo production were conducted in two experiments using reciprocal crosses. In the first, gametes from Bos taurus and Bos indicus were used; in the second, gametes from dairy and beef breeds of Bos taurus were used. In each experiment, both parental groups were used as sperm and oocyte donors, producing crossbred and purebred embryos. Oocytes obtained from abattoir-derived ovaries underwent in vitro maturation and in vitro fertilization with frozen semen. Embryos were cultured to blastocyst stage and observed. In the first experiment, higher (P < 0.05) rates of blastocyst formation were found for Bos taurus both as sires and as dams. Approximately 36% of the purebred Bos taurus oocytes and 21% of the purebred Bos indicus oocytes developed to blastocyst. Crosses averaged 16% resulting in a heterosis estimate of 45%. Ovaries from Bos indicus cows had more harvestable oocytes than did those from Bos taurus cows (P < 0.05). No evidence for heterosis was found for crosses within Bos taurus. Oocytes from beef cows had a higher rate of blastocyst formation than did those from dairy cows (30 vs. 24%, P < 0.05). These seemingly disparate results concerning heterosis were discussed in light of the period of genetic isolation of the parental populations in the two experiments.

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PubMed2000

Effect of osmotic stress on the developmental competence of germinal vesicle and metaphase II stage bovine cumulus oocyte complexes and its relevance to cryopreservation.

The effects of osmotic stress on germinal vesicle (GV) and metaphase II (MII) stage bovine cumulus oocyte complexes (COCs) were evaluated by first exposing them to various anisotonic NaCl solutions (75, 150, 600, 1200, 2400, and 4800 +/- 5 mOsm/kg) for 10 min and then returning them to isotonic TL-Hepes solution (270 +/- 5 mOsm/kg) at 20 +/- 2 degrees C. Percentages of oocyte maturation, fertilization, polyspermy, cleavage, and blastocyst formation were measured as endpoints. Exposure to anisotonic conditions had a significant (P < 0.05) effect on the developmental competence of both GV and bovine MII COCs. Oocytes at the GV stage were more sensitive to anisotonic stress than MII oocytes (P < 0.05). None of the GV oocytes developed to the blastocyst stage after exposure to hypertonic conditions (2400 or 4800 mOsm solutions), while exposure to hypotonic conditions (75 or 150 mOsm solutions) resulted in significantly lower (P < 0.05) blastocyst formation (9% and 13%, respectively) compared to the isotonic control (25%). A dramatic decrease to 4% development to blastocyst was observed for MII oocytes following exposure to a 4800 mOsm solution. Blastocyst formation of MII oocytes which were exposed to 75, 150, 600, 1200, or 2400 mOsm solutions were similar (15%, 20%, 18%, 14%, and 13%, respectively; P > 0.05), but lower (P < 0.05) than those in the control group (29%). Exposing GV oocytes to anisotonic conditions increased polyspermic fertilization (P < 0.05), although MII oocytes were not similarly affected (P > 0.05). These data support the hypothesis that osmotic stress is detrimental to bovine oocytes and must be considered when developing optimized cryopreservation procedures for these cells. Mol. Reprod. Dev. 55:212-219, 2000.

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PubMed1999

Efficacy of timed embryo transfer with fresh and frozen in vitro produced embryos to increase pregnancy rates in heat-stressed dairy cattle.

Our objective was to determine whether pregnancy rates in heat-stressed dairy cattle could be enhanced by timed embryo transfer of fresh (nonfrozen) or frozen-thawed in vitro-derived embryos compared to timed insemination. Ovulation in Holstein cows was synchronized by a GnRH injection followed 7 d later by PGF2 alpha and a second treatment with GnRH 48 h later. Control cows (n = 129) were inseminated 16 h (d 0) after the second GnRH injection. On d 7, a fresh (n = 133) or frozen-thawed (n = 142) in vitro-derived embryo was transferred to cows assigned for timed embryo transfer after categorizing the corpus luteum by palpation per rectum as 3 (excellent), 2 (good or fair), 1 (poor), and 0 (nonpalpable). Response to the synchronization treatment, determined by plasma progesterone concentration (ng/ml) < or = 1.5 on d 0 and > or = 2.0 on d 7, was 76.2%. Mean plasma progesterone concentration on d 7 increased as the quality of corpus luteum improved from category 0 to 3. Concentrations of progesterone in plasma were elevated (> or = 2.0 ng/ml) at 21 d in 64.7 (fresh embryo), 40.3 (frozen embryo), and 41.4 +/- 0.1% (timed insemination) of cows, respectively. Cows that received a fresh embryo had a greater pregnancy rate at 45 to 52 d than did cows that received a frozen-thawed embryo or timed insemination (14.3 > 4.8, 4.9 +/- 2.3%). Body condition (d 0) of cows influenced the pregnancy rate and plasma progesterone concentrations. In summary, timed embryo transfer with fresh in vitro-produced embryos in heat-stressed dairy cattle improved pregnancy rate relative to timed insemination.

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PubMed1998

Heterologous fertilization to characterize spermatozoa of the genus Bos.

Advances in assisted reproductive techniques, specifically, development of protocols for production of in vitro matured, fertilized and cultured domestic bovine embryos, offer opportunities to apply these techniques to nondomestic bovidae in species preservation. Domestic bovine oocytes were inseminated with nondomestic bovine spermatozoa. Effects of heparin concentration, sperm concentration and their interaction on total and normal in vitro fertilization rates and on subsequent embryo development were evaluated. In different replications, semen from 3 Bos bison, 2 Bos gaurus, 1 Bos grunniens, and 1 Bos javanicus bulls was used. Treatment of spermatozoa included 2 heparin levels (2 and 8 micrograms/mL) and 3 sperm concentrations (1, 3 and 5 x 10(6)/mL). The B. grunniens bull exhibited excessive polyspermy in all treatments; therefore, 1 replicate was completed using 2 levels of heparin (0 and 1 microgram/mL) and 2 sperm concentrations (1 and 2 x 10(6)/mL). After 18 to 22 h, cumulus cells were removed from presumptive zygotes, and a portion thereof was compressed between a slide and coverslip and fixed in acetic acid:ethanol solution. Light microscopy was used to visualize pronuclei and the second polar body as a determinant of fertilization. Remaining presumptive zygotes were placed into embryo culture medium, and blastocyst development was assessed on Days 7 and 8 (fertilization = Day 0). Percentages of total and normal fertilization and of blastocyst formation were analyzed by a logistic regression model, isolating effects due to bull, heparin and sperm concentration, and to their interaction. Work presented here suggests that, just as in Bos taurus, the nondomestic bulls in the Bos species seem to have individual heparin and sperm concentration requirements for successful IVF. We conclude that each bull, domestic or nondomestic, needs to be evaluated individually. Preliminary sperm characterization using domestic cattle oocytes would result in a greater potential for generating purebred embryos of the desired species should scarce female gametes become available.

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PubMed1998

Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos.

Data on biopsied, sexed and cryopreserved in vitro produced (IVP) bovine embryos, and their in vivo developmental competence are very limited. Two preliminary studies were conducted before the primary study. In Experiment 1, post-thaw in vitro developmental competence of biopsied and vitrified IVP embryos was evaluated using re-expansion as an endpoint. In Experiment 2, the pregnancy rates of biopsied fresh, frozen or vitrified embryos following single embryo transfer were compared. Since vitrified embryos resulted in a higher pregnancy rate than frozen-thawed embryos, in the primary study (Experiment 3), all IVP embryos were vitrified following biopsy and sexing (by DNA fingerprinting). In Experiment 3, we compared pregnancy initiation and calving results of heifers in the following treatments: 1) artificial insemination (AI); 2) AI plus contralateral transfer of a single embryo (AI + SET); 3) ipsilateral transfer of single embryo (SET); or 4) bilateral transfer of two embryos (DET). Birth weights, gestation lengths and dystocia scores were recorded. In Experiment 1, post-thaw re-expansion rate of biopsied and vitrified embryos was 85% (70/82). In Experiment 2, pregnancy rates (90 d) were 44% (7/16), 23% (3/13), and 50% (7/14) for vitrified, frozen and fresh embryos, respectively (P < 0.10). In Experiment 3, pregnancy rates of AI and SET were 65% (20/31) and 40% (16/40), respectively (P < 0.05). The pregnancy rate of AI + SET was 75% (27/36) with 11 carrying twins, and the pregnancy rate of DET was 72% (26/36) with 10 carrying twins. All AI fetuses were carried to term, but only half the SET fetuses were carried to term. Similar calving rates were observed in the AI + SET and DET groups, 76 and 70%, respectively, of those pregnant at Day 40. Mean birth weight, dystocia score and gestation length of AI calves were not different from those of SET calves. Mean birth weight and dystocia score of single-born calves were greater than those of twin born calves (P < 0.05). These data demonstrate that biopsied IVP bovine embryos can be successfully cryopreserved by vitrification and following post-thaw embryo transfer, acceptable rates of offspring with normal birth weights can be obtained without major calving difficulties.

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PubMed1998

Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation lengths.

In vitro and in vivo developmental competence of fresh and cryopreserved in vitro produced (IVP) bovine embryos was evaluated up to birth. Three experiments were done. The objective in the first experiment was to develop an optimal vitrification procedure for IVP bovine embryos by determining effects of exposure time (2, 5, 10, 20 min) and temperature (4, 22, 27 degrees C) in cryoprotective agents prior to vitrification on their post-thaw viability. The best combination was used in Experiments 2 and 3. In the second experiment, the importance of post-thaw morphologic selection on pregnancy rates was determined by transferring either selected or unselected single embryos. In the third experiment, pregnancy initiation, maintenance and calving results of vitrified embryos were compared with fresh and conventionally frozen embryos. Fetal losses, birth weights, gestation lengths and frequency of dystocia in the third experiment were monitored. The interaction of exposure time and temperature on both post-thaw re-expansion and hatching rates was significant (P < 0.01). Five minute exposure at 27 degrees C was optimal. In the second experiment, post-thaw selected vitrified embryos had higher pregnancy rates than unselected embryos (P < 0.05). In the third experiment, the pregnancy rate of vitrified embryos did not differ from that of fresh embryos (P > 0.05). However, pregnancy rate of conventionally frozen embryos was lower than that of fresh or vitrified embryos (P < 0.05). Of 92 calves born, 53 were male and 39 were female. Birth weights and dystocia scores of single-born calves did not differ between sexes (P > 0.05). Twin-born calves were lighter than single-born calves (P < 0.05). Overall, the data demonstrate that the transfer of vitrified IVP bovine embryos can result in healthy, apparently normal calves similar to those derived from transfer of fresh and conventionally frozen IVP bovine embryos.

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PubMed1994

Oocyte maturation, fertilization and embryo development in vitro and in vivo in the gaur (Bos gaurus).

A study was conducted to evaluate the potential of rescuing immature oocytes from the ovaries of an endangered wild bovid, the gaur (Bos gaurus). Recovered, immature gaur oocytes (n = 59) placed in culture were evaluated for: (1) nuclear maturation after 22 h of culture, (2) fertilization with either thawed homologous (gaur) or heterologous (Bos taurus) spermatozoa 18 h after insemination and (3) embryo development. Gaur oocytes (n = 6) evaluated by fixation and staining at 22 h had all matured to metaphase II in vitro. Insemination of gaur oocytes in vitro resulted in normal fertilization (defined as the presence of spermatozoa head or two pronuclei) and embryo development to the two- and four-cell stage of 53.6% (15 of 28) and 50.0% (9 of 18), respectively, using homologous spermatozoa. The incidence of normal fertilization of in vitro matured (IVM) gaur oocytes with heterologous spermatozoa was 53.8% (7 of 13). Insemination of domestic cow oocytes in vitro resulted in normal fertilization and embryo development of 41.7% (45 of 108) and 60.0% (12 of 20), respectively, using heterologous spermatozoa. Two of four gaur embryos (50%) developed to the blastocyst stage by day 7. Embryo transfer of these two conspecific gaur blastocysts into two Holstein recipients resulted in one confirmed pregnancy. One live-born calf was delivered by Caesarean section 308 days after embryo transfer. These results demonstrate the potential of combined IVM and IVF for recovering immature germplasm from an endangered species. Specifically, immature gaur ovarian oocytes are capable of in vitro maturation and fertilization with thawed homologous spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

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