Biomedical subjects

R J Mapletoft

Publications and source records attributed to R J Mapletoft.

At least 37 records · Page 2Linked to original sources

PubMed2000

The effect of macromolecular supplementation on the surface tension of TCM-199 and the utilization of growth factors by bovine oocytes and embryos in culture.

The objectives of this study were to determine the surface tension of bovine follicular fluid (BFF) and TCM-199, and the effects of the synthetic surfactant, Twin-80, or FCS, in TCM-199 on surface tension measurements and subsequent effects on bovine oocyte and embryo development. The surface tension of BFF was determined to be approximately 45.5 mN m(-1) at 25 degrees C and approximately 42.7 mN m(-1) at 39 degrees C, which was comparable to the surface tension of TCM-199 containing Twin-80 (45.7 and 43.2 mN m(-1), respectively). There was no difference in surface tension measurements of BFF from follicles 2-7 mm or 8-15 mm in diameter. Both Millipore water and phosphate buffered saline (PBS) had a surface tension measurement of 69.5 mN m(-1) at 39 degrees C. Although the presence of Twin-80 in TCM-199 resulted in a reduction in surface tension measurement as compared with unsupplemented TCM-199, there was no effect on the number of oocytes reaching metaphase II. However, the addition of epidermal growth factor (EGF) to TCM-199 containing Twin-80 did result in increased maturation rates of oocytes in vitro. There was no effect of insulin, transferrin, and selenium (ITS), or EGF on surface tension measurement of TCM-199, but significantly more zygotes cleaved and developed to morulae/blastocysts in TCM-199 containing both Twin-80 and growth factors. The addition of 20 microg EGF ml(-1) to TCM-199 containing Twin-80 was as efficacious in supporting bovine embryos in culture as was the addition of 5% or 10% fetal calf serum. This study demonstrates that surface-active components in culture media positively affect bovine oocyte maturation and embryo development in culture. Data also suggest that non-ionic surfactants, such as Twin-80 in TCM-199, may successfully replace the surface-active properties but not the embryotrophic properties of serum in embryo maturation/culture media. Although commercial TCM-199 (containing Twin-80) did not require the addition of other surface-active compounds to lower surface tension, it did benefit from the addition of growth promoting factors, which were also provided by serum.

Animals↗

PubMed2000

Effect of reduced concentrations of glycerol and various macromolecules on the cryopreservation of mouse and cattle embryos.

The effect of different macromolecules [bovine serum albumin (BSA), Pluronic F-68, (ET surfactant), or sodium hyaluronate (SH)] on postthaw survival of mouse morulae and in vivo- and in vitro-derived bovine blastocysts frozen in 10, 5, or 1% glycerol solutions was investigated. Embryos were equilibrated with cryoprotectant solution at 25 degrees C for 10 min, seeded at -5 degrees C, cooled at 0.5 degrees C/min to -35 degrees C, and plunged into liquid nitrogen. Embryos were thawed in a 35 degrees C water bath, glycerol was removed with 0.6 M sucrose at 25 degrees C for 5 min, and postthaw viability was evaluated after 1, 24, and 48 h in culture. The addition of BSA supplementation improved postthaw survival of mouse morulae frozen in 5% glycerol, but not in 10% glycerol. All three macromolecular supplements were effective in increasing survival of mouse morulae in 5% glycerol but only BSA and SH were effective in increasing postthaw survival of in vivo- and in vitro-derived bovine blastocysts. None of the macromolecular supplements improved postthaw survival of embryos frozen in 1% glycerol.

Animals↗

PubMed2000

Estrus synchronization and pregnancy rates in beef cattle given CIDR-B, prostaglandin and estradiol, or GnRH.

Two experiments were conducted to determine estrous response and pregnancy rate in beef cattle given a controlled internal drug release (CIDR-B) device plus prostaglandin F2 alpha (PGF) at CIDR-B removal, and estradiol or gonadotropin releasing hormone (GnRH). In Experiment I, crossbred beef heifers received a CIDR-B device and 1 mg estradiol benzoate (EB), plus 100 mg progesterone (E + P group; n = 41), 100 micrograms gonadotropin releasing hormone (GnRH group; n = 42), or no further treatment (Control group; n = 42), on Day 0. On Day 7, CIDR-B devices were removed and heifers were treated with PGF. Heifers in the E + P group were given 1 mg EB, 24 h after PGF, and then inseminated 30 h later. Heifers in the GnRH group were given 100 micrograms GnRH, 54 h after PGF, and concurrently inseminated. Control heifers were inseminated 12 h after onset of estrus. The estrous rate was lower (P < 0.01) in the GnRH group (55%) than in either the E + P (100%) or Control (83%) groups. The mean interval from CIDR-B removal to estrus was shorter (P < 0.01) and less variable (P < 0.01) in the E + P group than in the GnRH or Control groups. Pregnancy rate in the E + P group (76%) was higher (P < 0.01) than in the GnRH (48%) or Control (38%) groups. In Experiment II, 84 cows were treated similarly to the E + P group in Experiment I. Cows received 100 mg progesterone and either 1 mg EB or 5 mg estradiol-17 beta (E-17 beta) on Day 0 and either 1 mg of EB or 1 mg of E-17 beta on Day 8 (24 h after CIDR-B removal), in a 2 x 2 factorial design, and were inseminated 30 h later. There were no differences among groups for estrous rates or conception rates. The mean interval from CIDR-B removal to estrus was 44.2 h, s = 11.2. Conception rates were 67%, 62%, 52%, and 71% in Groups E-17 beta/E-17 beta, E-17 beta/EB, EB/E-17 beta, and EB/EB, respectively. In cattle given a CIDR-B device and estradiol plus progesterone, treatment with either EB or E-17 beta effectively synchronized estrus and resulted in acceptable conception rates to fixed-time artificial insemination.

Animals↗

PubMed2000

A study of the inheritance of a bleeding disorder in Simmental cattle.

A study was designed to determine the inheritance pattern of a blood platelet aggregation disorder in Simmental cattle utilizing embryo transfer technology. A Simmental donor cow that had previously produced a calf with the platelet aggregation disorder was superovulated and mated to a bull that had also produced affected offspring. Twenty-seven calves were produced from the 63 (42.9%) embryos transferred. This somewhat lower than expected pregnancy rate is suggestive of an increased rate of embryo loss. Twenty-three of 25 (92%) calves had normal platelet aggregation patterns and 2 failed to show any evidence of platelet aggregation. Data are suggestive that inheritance is not simple Mendelian recessive. A more likely scenario is that the defect is the result of the inheritance of at least 2 genes, which is also consistent with the sporadic incidence reported in the population at large.

Animals↗

PubMed1999

Effect of LH or GnRH on the dominant follicle of the first follicular wave in beef heifers.

A study was designed to characterise ovarian follicular dynamics in heifers treated with porcine luteinizing hormone (pLH) or gonadotropin releasing hormone (GnRH) on days 3, 6 or 9 (ovulation = day 0), corresponding to the growing, early-static, and late-static phases of the first follicular wave. Following ovulation, 65 beef heifers were assigned, by replicate, to the following seven treatment groups: 25 mg im of pLH on days 3, 6 or 9 (n = 9 per group); 100 microg im of GnRH on days 3, 6 or 9 (n = 9 per group); or controls (no treatment; n = 11). Ovulation occurred within 36 h in 67%, 100% and 67% of heifers treated with pLH and in 89%, 56% and 22% of heifers treated with GnRH on days 3, 6 or 9, respectively (treatment-by-day interaction, P < 0.09). Combined for all treatment days, ovulation rates were 78% and 56% in pLH- and GnRH-treated groups, respectively (P < 0.09). Overall, mean day (+/- SD) of emergence of the second follicular wave in heifers that ovulated was different from that in controls or in heifers that did not ovulate (P < 0.05). Mean (+/- SD) day of emergence of the second wave occurred earlier (day 5.6+/-1.2; P < 0.05) in heifers that ovulated after treatment on day 3 (n = 14) than in controls (day 8.7+/-1.6; n = 11); however, wave emergence in all heifers treated on day 6 (day 8.1+/-0.5; n = 18) did not differ from controls, regardless of whether or not ovulation occurred. In the heifers that ovulated in response to treatment on day 9 (n = 8), the emergence of the second follicular wave was delayed (day 10.9+/-0.4; P < 0.05). The day of emergence of the second wave in the 14 treated heifers that failed to ovulate, irrespective of the day of treatment (day 8.9+/-1.4) did not differ from control heifers. The emergence of the second wave was more synchronous in day 6 heifers (regardless of whether they ovulated) and in day 9 heifers that ovulated compared to control heifers (P < 0.05). Results did not support the hypothesis that the administration of pLH or GnRH at known stages of the follicular wave in cycling heifers would consistently induce ovulation or atresia and, thereby, induce emergence of a new follicular wave at a predictable interval. New wave emergence was induced consistently (1.3 days post-treatment) only in those animals that ovulated in response to treatment. However, 22% of LH-treated heifers and 44% of GnRH-treated heifers failed to ovulate. Treatments did not induce atresia of the dominant follicle or alter the interval to new wave emergence in animals that did not ovulate in response to treatment.

Animals↗

PubMed1999

An investigation of the fertilizing characteristics of pyriform-shaped bovine spermatozoa.

Two breeding trials were done to determine the effect of pyriform-shaped bovine spermatozoa on fertility. In breeding trial (1), heifers were superstimulated by injecting follicle-stimulating hormone (FSH) and randomly allotted to an affected Bull A (n = 21) or a Control Bull, C (n = 18). Semen from Bull A contained 85% pyriform spermatozoa while that from the Control Bull C contained 91% normal sperm and 2% pyriform spermatozoa. Fertilization rate was lower (P = 0.01) for Bull A (total of 63 ova/embryos; 68.5%) than for the Control Bull, C (total of 81 ova/embryos; 84.4%). In breeding trial (2), 37 oestrus-synchronized heifers were randomly allotted to Bull A (n = 19) or Bull C (n = 18). Pregnancy rates at Day 60 (37% and 61% for Bulls A and C, respectively; P = 0.22) and rate of embryo/fetal loss between Days 22 and 60 of pregnancy (23% and 8% for Bulls A and C, respectively; P = 0.55) were not different. In vitro experiments involving the same Bull A and another Control Bull, B, were designed to explain the mechanism of infertility caused by pyriform sperm. The mean (+/- SEM) number of sperm bound to the zona pellucida was lower (P < 0.05) for Bull A (24.6+/-1.2) than for the Control Bull, B (46.6+/-1.9) and the percentage of zonae penetrated by sperm from Bull A (56.0%) and Bull B (82.8%) was also different (P < 0.05). The percentage of pyriform sperm from Bull A bound to (53%), and penetrating (49%) the zona pellucida was lower (P < 0.05) than that in the inseminate (85%). Although fertilization rates (64.1% and 72.8%) were not different (P = 0.5), cleavage rates (48.2% vs. 74.1%) and morula production rates (24.8% vs. 37.7%) were less (P < 0.05) for Bull A than for Bull B, respectively. In summary, pyriform sperm had reduced capability to bind to and penetrate the zona pellucida, and zygotes (resulting from the fertilization of oocytes by pyriform sperm) appeared to have a reduced ability to initiate cleavage.

Animals↗

PubMed1999

Changes in the cumulus-oocyte complex of subordinate follicles relative to follicular wave status in cattle.

We investigated factors that affect cumulus-oocyte complex (COC) morphology and oocyte developmental competence in subordinate follicles on different days after follicular wave emergence in beef heifers. In Experiment 1, heifers (n = 13) were assigned at random to COC aspiration during the growing/static (Days 1 to 3) or regressing (Day 5) phase of subordinate follicle development (follicular wave emergence = Day 0). Follicular wave emergence was induced by transvaginal ultrasound-guided follicular ablation, ovaries were collected at slaughter, all follicles > or = 2 mm except the dominant follicle were aspirated, and COC were microscopically evaluated for morphology. There was a greater percentage of COC with expanded cumulus layers on Day 5 (42.4%) than on Days 1 to 3 (2.2%). In Experiment 2, heifers (n = 64) at random stages of the estrous cycle had all follicles > or = 5 mm ablated and 4 d later, 2 doses of PGF were injected 12 h apart; heifers were monitored daily by ultrasonography for ovulation (Day 0 = follicular wave emergence). Heifers were assigned to the following time periods for oocyte collection from subordinate follicles: Days 0 and 1 (growing phase), Days 2, 3 and 4 (static phase), and Days 5 and 6 (regressing phase). Ovaries were individually collected at slaughter, and all follicles > or 2 mm except for the dominant follicle were aspirated. The COC were morphologically evaluated and then matured, fertilized and cultured in vitro. Expanded COC were more frequent during the regressing phase (53.4%) than the growing or static phase (14.4 and 17.8%, respectively; P < 0.05). While the proportions of COC with > or = 4 layers of cumulus cells and denuded oocytes were higher (P < 0.05) in the growing and static phases, the production of morulae was highest (P < 0.05) with COC collected from subordinate follicles during the regressing phase. In Experiment 3, heifers (n = 18) were assigned at random to oocyte collection from subordinate follicles 3 and 4 d (static phase) or 5 and 6 d (regressing phase) after follicular wave emergence. The heifers were monitored ultrasonically for ovulation (Day 0 = follicular wave emergence); COC were collected from all follicles (> or = 5 mm) except for the dominant follicle by transvaginal ultrasound-guided follicle aspiration 3 to 6 d later. Recovered oocytes were stained and examined microscopically to evaluate nuclear maturation. A higher proportion of oocytes collected on Days 5 and 6 showed evidence of nuclear maturation (50%) than on Days 3 and 4 (8.3%; P < 0.05). Results support the hypothesis that COC morphology and oocyte developmental competence change during the growing, static and regressing phases of subordinate follicle development.

Animals↗

PubMed1999

Synchronization of estrus in beef cattle with norgestomet and estradiol valerate.

Fifty-six cows received a norgestomet implant and an injection of norgestomet and estradiol valerate; half (n = 28) received 500 IU equine chorionic gonadotrophin (eCG) at implant removal, 9 d later. A third group (n = 25) received 2 doses of cloprostenol (500 micrograms) 11 d apart. Estrous rate was higher (P < 0.05) for cows given norgestomet and estradiol plus 500 IU eCG (75.0%) than for those receiving cloprostenol (44.0%); for those receiving norgestomet and estradiol alone, it was intermediate (67.8%). Pregnancy rates to artificial insemination (after estrus or timed) were higher (P < 0.05) for cows given norgestomet and estradiol than for those given cloprostenol (23 of 28, 82.1% vs 13 of 25, 52.0%), and intermediate (67.8%) for those given norgestomet and estradiol plus eCG. In a second experiment, for heifers treated with norgestomet and estradiol plus eCG (n = 15) or with 2 doses of cloprostenol (n = 16), estrous rates were 66.7% vs 56.2% (P > 0.5), ovulation rates were 100.0% vs 81.2% (P = 0.08), intervals from implant removal or cloprostenol treatment to estrus were 48.0 +/- 4.4 hours vs 61.3 +/- 7.0 hours (P = 0.12) and to ovulation were 70.4 +/- 4.4 hours vs 93.2 +/- 7.5 hours (P < 0.01), respectively; pregnancy rates were 41.7 and 35.7%, respectively (P > 0.5). Norgestomet and estradiol were as good as (heifers) or superior to (cows) a 2-dose cloprostenol regimen. In cows given norgestomet and estradiol, injecting eCG at implant removal did not significantly improve estrous or pregnancy rates.

Analysis of Variance↗

PubMed1998

Fertilization characteristics and in vitro embryo production with bovine sperm containing multiple nuclear vacuoles.

An in vitro fertilization and culture system was used to determine the effect of multiple nuclear vacuoles in bovine spermatozoa on fertilization and early embryonic development. After swim-up, semen parameters were similar between 2 bulls except that 60% of spermatozoa from bull A contained multiple nuclear vacuoles, whereas no spermatozoa from bull B (control) contained vacuoles. In Experiment 1, in vitro-matured (IVM) oocytes were inseminated with frozen-thawed semen from the 2 bulls to determine the ability of vacuolated sperm to bind with the zona pellucida. The mean number of spermatozoa bound to the zona pellucida was less (P < 0.05) for bull A (85.7 +/- 5.7; n = 112) than for bull B (108.9 +/- 5.4; n = 130). In Experiment 2, the percentages of zonae penetrated by spermatozoa from bull A (151 of 201; 75%) and bull B (116 of 150; 77%) were not different. However, the percentages of vacuolated spermatozoa from bull A bound to (43%) and penetrating the zona pellucida (34%) were lower than those in the inseminate (60%). In Experiment 3, fertilization rates, as evidenced by the presence of two pronuclei, were not different for bull A (101 of 136; 74%) and bull B (89 of 115; 77%). In Experiment 4, there was no significant difference in percentage cleavage (72.1% versus 76%) and morulae (29.2% versus 34.8%) or blastocyst production (7.2% versus 8.4%) for bulls A and B, respectively. Data suggest that spermatozoa with multiple nuclear vacuoles are defective in zona binding. However, vacuolated spermatozoa gaining access to the ooplasm apparently participate in fertilization and early embryonic development.

Animals↗

PubMed1998

Ovarian follicular responses in dairy cows treated with GnRH and cloprostenol.

Lactating, nonpregnant (with a corpus luteum) Holsteins were given 100 ug GnRH (n = 12) or saline (n = 12) and 500 ug cloprostenol 6 d later. Following luteolysis, ovulation occurred 10.1 +/- 0.2 d (range, 9-12 d) after GnRH and 8.6 +/- 1.0 d (range, 3-12 d) after saline (differences between groups: means, P > 0.05; variability, P < 0.001). Treatment with GnRH and cloprostenol resulted in a relatively synchronous ovulation.

Animal Husbandry↗

PubMed1998

The effect of estradiol cypionate (ECP) on ovarian follicular development and ovulation in dairy cattle.

The efficacy of estradiol cypionate (ECP) for synchronizing ovarian follicular development was determined in lactating Holstein-Friesian cattle. In Experiment 1, 13 cattle were given simultaneous intramuscular (i.m.) injections of 100 mg progesterone and 0 (control), 0.5 or 1.0 mg ECP on Day 3, after a synchronized ovulation (Day 0). Maximum diameter of the dominant follicle of Wave 1 was significantly larger in control cattle than in those given 0.5 or 1.0 mg ECP (means: 15.7, 13.2, and 12.9 mm, respectively). Mean day of emergence of Wave 2 was significantly later in controls than in those given 1.0 mg ECP, with the 0.5 mg group intermediate (Days 10.2, 8.8 and 9.5, respectively). In Experiment 2, 14 cattle were given a CIDR-B and IM injections of 1 mg ECP and 50 mg progesterone without regard to stage of cycle (treatment = Day 0). On Day 8, the CIDR-B was removed and 500 micrograms cloprostenol injected, IM. Mean days of wave emergence (Day 3.4; range: -2 to 7) and ovulation (Day 12.1; range: 10 to 14) indicated that ECP had limited efficacy for synchronizing follicular development and ovulation in dairy cattle when given at random stages of the estrous cycle.

Animal Husbandry↗

PubMed1997

Vitrification of bovine IVF blastocysts in an ethylene glycol/sucrose solution and heat-stable plant-extracted proteins.

The use of heat-stable plant proteins in an ethylene glycol-based solution for the vitrification of in vitro-derived embryos was examined. Day 7, 8 and 9 bovine in vitro matured, fertilized and cultured (IVMFC), full and expanded blastocysts were vitrified in solutions composed of 40% ethylene glycol (EG) plus 0.3 M sucrose supplemented with 20% Ficoll and 0.3% BSA (VF-1), 25 mg/ml heat-stable plant proteins (HSPP; VF-2), or with no supplement (VF-3). In Experiment 1, embryos were expelled from the straw after thawing, and EG was diluted from embryos with 0.5 M sucrose. There were no differences in post-thaw embryo survival rates or in hatching/hatched rates after 24 h of culture between the VF-1, VF-2 and VF-3 solutions (40.1, 54.1 and 50.8% and 10.7, 16.4 and 17.5%, respectively). Transfer of 12 frozen/thawed embryos to 6 recipients (2 recipients per treatment) resulted in 2 pregnancies from the VF-2 group and 1 pregnancy from the VF-3 group. In Experiment 2, EG was diluted from embryos after thawing within the straw with 0.5 M sucrose. There were no differences in post-thaw survival or hatching/hatched rates after 24 h of culture (19.0, 13.6 and 23.8% and 9.5, 9.0 and 14.4% for VF-1, VF-2 and VF-3, respectively). Transfer of 6 frozen/thawed embryos to 3 recipients (1 recipient per treatment) resulted in no pregnancies. The post-thaw histology of Day 7, 8 and 9 IVMFC blastocysts showed typical ultrastructure with well preserved cell-to-cell contacts. There were no major differences in the fine structure of blastocysts regardless of treatment. The use of HSPP at a concentration of 25 mg/ml in the vitrification medium did not affect the post-thaw embryo survival over that of no protein supplementation. The presence of macro molecules in a 40% EG/sucrose vitrification solution also did not improve post-thaw viability of IVMFC-derived blastocysts.

Journal Article↗

PubMed1997

Superovulatory response following ablation-induced follicular wave emergence at random stages of the oestrous cycle in cattle.

Based on the premise that superovulation in cattle is optimal when superstimulation is initiated at the time of follicular wave emergence, the present study was done in beef heifers to determine if the superovulatory response following a single bolus of gonadotrophin treatment after follicle ablation (induced wave) at random stages of the oestrous cycle is comparable to the same gonadotrophin treatment at mid-dioestrus (spontaneous wave). In Experiment 1, heifers were assigned to nonablation (n = 18) and ablation (n = 20) groups. In nonablated heifers, superstimulatory treatment was given as a single subcutaneous injection (Folltropin-V, 400 mg) at mid-di-oestrus to coincide with emergence of the spontaneous follicular wave 8 to 12 days after oestrus. In ablated heifers, the same superstimulatory treatment was given 1 day after ablation of all follicles > or = 5 mm at random stages of the oestrous cycle to coincide with emergence of the ablation-induced wave. In both the nonablation and ablation groups, PGF2 alpha (Estrumate, 500 micrograms) was given 48 h after the superstimulatory treatment and artificial insemination was done 60 and 72 h later. Reproductive tracts were collected at the time of slaughter 6 or 7 days after insemination. Observations made in Experiment 1, indicated that some ablated heifers had only partial luteal regression at the time of insemination, while some others exhibited behavioral oestrus as early as 24 h after PGF2 alpha treatment. The design was amended in Experiment 2 to address these problems. Heifers were assigned to nonablation (n = 17), ablation-alone (n = 20) or ablation plus progestogen (n = 20) groups. Follicle ablation, superstimulatory treatment, artificial insemination and collection of reproductive tracts were done as in Experiment 1. However, all heifers were given two doses of PGF2 alpha (500 micrograms/dose) 48 and 60 h after superstimulatory treatment to ensure complete luteal regression, and heifers in the ablation plus progestogen group received a norgestomet ear implant at the time of follicle ablation to prevent early ovulations. The implant was removed at the time of the second PGF2 alpha treatment. In Experiments 1 and 2, the means for the ovarian and superovulatory responses were not significantly different between groups. Averaged over the nonablation and all ablation groups for Experiments 1 and 2, the mean number. of corpora lutea, fertilized ova and transferable embryos were 22.9 vs 18.6, 7.3 vs 7.8 and 5.4 vs 5.6, respectively. In summary, follicle ablation at random stages of the oestrous cycle followed by a single bolus of gonadotrophin treatment 1 day later resulted in a superovulatory response that was comparable to the same superstimulatory treatment administered around the time of spontaneous wave emergence at mid-dioestrus. The ablation/superstimulation method described herein offers the advantage of initiating superstimulatory treatment forthwith and assuring that treatment is concomitant with wave emergence to achieve an optimal superovulatory response. Moreover, the full extent of the oestrous cycle is available for superstimulation and the need for detecting oestrus or ovulation and waiting 8 to 12 days to initiate treatment is eliminated.

Animals↗

PubMed1996

Estrus synchronization in cattle using estradiol, melengestrol acetate and PGF.

In Experiment 1, all cattle were fed MGA (0.5 mg/head/d) for 7 d (designated Days 0 to 6) and given PGF on Day 6. One-half were administered estradiol valerate (EV; 5 mg, im) on Day 0. At Location 1, a higher proportion (P < 0.005) of EV-treated heifers were detected in estrus and bred by AI between Days 7 and 13 than control heifers not receiving EV (27 of 33 versus 15 of 32), but the number of pregnancies (12 vs 10) was not significantly different. Eighty-three of 104 EV-treated and 89 of 106 control cows were inseminated, resulting in 50 and 45 pregnancies, respectively (not significant). At Location 2, cattle were similarly treated and exposed to bulls on Days 7 to 49. Fall pregnancy rate was higher (P < 0.015) for EV-treated than control heifers (44 of 48 vs 33 of 46), but was not significantly different for cows (22 of 26 vs 19 of 23). In Experiment 2, estradiol 17beta (E17beta; 5 mg, im) and progesterone (100 mg, im) were administered on Day 0 (instead of EV). In a third group (designated the PGF group), cattle were bred on Days 0 to 6, and PGF was administered on Day 6 to those not yet bred. For 213 cows, the percentage pregnant to a synchronized estrus was greater in the PGF group (72%) than in either the control group treated with MGA (49%; P = 0.005) or the group receiving MGA and E17beta (54%; P < 0.025). Fall pregnancy rates were 91, 89, and 96% for the 213 cows (not significant) and 89, 93, and 98% for 131 heifers (not significant) in the PGF, MGA and E17beta groups, respectively. In cattle without a functional CL, the average diameter of the largest follicle at Day 6 was 1 to 2 mm smaller in the E17beta + MGA group than in the MGA group (difference significant only in cows at Location 1). Combined for both locations, the synchronized pregnancy rate in heifers without a functional CL on Day 6 was higher (P < 0.05) in the E17beta + MGA group than in the MGA group (11 of 21, 52% versus 4 of 20, 20%). Estrogen treatment caused regression of ovarian follicles with emergence of a new follicular wave. Including estrogen in an estrus synchronization program utilizing MGA and PGF significantly increased fall pregnancy rate in heifers (at 1 location) and the synchronized pregnancy rate of heifers without a functional CL at the time of PGF treatment (combined for both locations).

Journal Article↗

PubMed1996

Effect of progestogen plus estradiol-17beta treatment on superovulatory response in beef cattle.

A series of 3 experiments were conducted to evaluate superovulatory response following exogenously controlled follicular wave emergence in cattle. In Experiment 1 the hypothesis was tested that treatments with progestogen plus estradiol-17beta (E-17beta) would result in the emergence of a wave of ovarian follicles that are as responsive to exogenous gonadotropins as those of a spontaneous follicular wave. Beef cows and heifers either received a progestogen ear implant on Day 0 (ovulation) plus 5 mg im E-17beta on Day 1 and were superstimulated on Day 5, or did not receive implants but were superstimulated on Day 8 (expected day of emergence of the second follicular wave). The cattle received 400 mg NIH-FSH-P1 of Folltropin-V, given in a single subcutaneous injection or twice daily as intramuscular injections over 4 d. No significant differences were detected between the 2 groups in the number of corpora lutea (CL), ova/embryos collected, fertilized ova and transferable embryos. In Experiment 2 superstimulatory responses to a single subcutaneous injection of Folltropin-V were compared between heifers in which follicle wave emergence was synchronized with progestogen plus E-17beta at unknown stages of the estrous cycle with those treated following a conventional method of superstimulation at middiestrus. Superstimulation 4 d after E-17beta treatment in heifers with progestogen implants resulted in a similar superovulatory response and higher fertilization rates than those initiated 8 to 12 d after estrus. In Experiment 3 the ovarian response to a single- versus multiple-injection superstimulatory treatment protocol was compared in heifers given progestogen plus E-17beta to induce synchronous wave emergence. The number of CL, ova/embryos collected, fertilized ova and viable embryos were not different between groups. Superstimulatory treatments initiated 4 d after E-17beta treatment of cattle with progestogen implants resulted in comparable ovulatory responses to treatments initiated at the time of spontaneous wave emergence or during middiestrus. Synchronizing wave emergence in a group of randomly cycling cattle obviated the need of estrus detection and synchronization prior to superstimulation.

Journal Article↗

PubMed1996

Survival rates and sex ratio of bovine IVE embryos frozen at different developmental stages on day 7.

Two experiments were designed to determine the effects of stage of development on Day 7 of in vitro-produced bovine embryos on survival after deep freezing and on sex ratio. Bovine IVF embryos and bovine oviductal epithelial cells (BOEC) were co-cultured in TCM-199 and, on Day 7 after insemination (Day 0), were morphologically evaluated and divided into groups by developmental stage. In Experiment 1, embryos classified as early blastocysts, blastocysts and full-expanding blastocysts were randomly subdivided into 2 groups by replicate: 50% of the embryos were placed immediately in a new BOEC co-culture (fresh group), while the other 50% were frozen, thawed and placed in a new BOEC co-culture (frozen/thawed group). Embryos were frozen in 1.5 M glycerol using a standard slow cooling technique. Fresh and frozen/thawed embryos were compared for survival rate (embryos hatching/hatched) in BOEC co-culture over the following 3 d (i.e., Days 7 to 10). The overall survival of the 425 embryos (early to full-expanding blastocysts) was 33% and was not different between fresh (35%) and frozen/thawed (30%) embryos. Survival of embryos cultured fresh or after freezing/thawing was higher for full-expanding blastocysts than for early blastocysts or for blastocysts, both of which were not different. In Experiment 2, all frozen/thawed embryos used in Experiment 1 plus all morulae and hatched blastocysts collected and frozen on Day 7 without regard to survival were sexed utilizing the polymerase chain reaction (PCR) technique. Sex of the embryos, by stage of development on Day 7, was determined in order to compare the rate of development in BOEC co-culture with the sex ratio (percentage of males). A total of 235 embryos was sex-determined with an overall percentage of males of 51%, which was not different from the expected 1:1 sex ratio. Both full-expanding blastocysts and hatched blastocysts had a significantly higher (P < 0.05) proportion of males (68 and 100%, respectively), while morulae had a significantly lower proportion of males (24%). Early blastocysts and blastocysts did not differ from a 1:1 sex ratio. The results indicate that male embryos develop faster in vitro than female embryos. The higher survival rate of full-expanding blastocysts after freezing/thawing, and the production of a higher number of males than females among embryos of this developmental stage suggest that a greater number of male fetuses may result from the successful freezing and transfer of in vitro-produced bovine embryos.

Journal Article↗

PubMed1996

Cryopreservation of mammalian embryos and oocytes: recent advances.

The cryopreservation of embryos of most domestic species has become a routine procedure in embryo transfer, and recently, advances have been made in the cold storage of mammalian oocytes. The ability to sustain viable oocytes and embryos from mammalian species at low temperature for prolonged periods of time has important implications to basic and applied biotechnology. Recent advances in the study of physico-chemical behaviour of different cryoprotectants, use of various macromolecule additives in cryoprotective solutions and isolation and use of proteins of plant and animal origin with antifreeze activity offers many new options for cryopreservation of oocytes and embryos of animal and human origin. At the same time rapidly developing methods of oocyte/embryo manipulation such as in vitro embryo production, embryo splitting, embryo biopsying for gene and sex determination, embryo cloning and the isolation of individual blastomers, create new challenges in cryopreservation. Very recent advances in the cryopreservation of mammalian oocytes, in vivo- and in vitro-derived embryos, and micromanipulated embryos are reviewed in this manuscript.

Journal Article↗

PubMed1995

Morphology and location of attached follicular cumulus-oocyte complexes in horses, cattle and llamas.

Morphology and location of the attached cumulus-oocyte complex (COC) were studied in slaughter-house ovaries in horses (49 follicles, 9 to 44 mm), cattle (68 follicles, 6 to 18 mm), and llamas (38 follicles, 3 to 14 mm). The expected point of ovulation was marked, using the ovulation fossa in mares and the center of the projecting follicular surface in cattle and llamas. A follicle was dissected from an ovary, and tissue was removed from the follicle until the COC became visible by transillumination. However, most llama follicles protruded prominently from the ovarian surface so that dissection was not required to locate the COC. The COC was more readily recognized from the external follicular surface in mares and llamas than in cattle, primarily because of a dark oocyte. Compact COC's projected into the antrum with a smooth dome-shape in horses. The COC's in cattle were also dome-shaped but were more irregular and a few contained prominent processes. The mean diameter of the isolated follicle was calculated from 3 planes, except that in llamas the follicles were spherical so that the 3 dimensions were identical. The angle between a straight line connecting the expected ovulation site and the opposite pole and a straight line from the ovulation site to the COC was defined as the COC-location angle. This angle was chosen because it is unaltered by size of a sphere (45 degrees for a COC at the equator). The mean (+/-SEM) COC-location angle differed (P < 0.01) among horses (39.9 +/- 3.3), cattle (50.0 +/- 2.5), and llamas (64.8 +/- 2.1). In mares, the locations of the COC's did not differ from equality between follicular hemispheres, but in cattle and llamas the COC's were located with greater frequency (P < 0.05) in the hemisphere containing the expected ovulation site (cattle, 65%; llamas, 91%).

Journal Article↗