Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser och 2 appendix.

Experiments were initiated to determine whether exogenous estradiol-17beta (E2) and oxytocin (OT) can be used to dilate the cervix and improve transcervical embryo transfer (ET) procedures for sheep. However, there was concern that the E2-OT treatment may alter luteal function and that embryo quality would decrease as the superovulatory response to FSH increased. In Exp. 1, 32 ewes were assigned to a 2 x 2 factorial array of treatments. On d 7, ewes received an i.v. injection of either 100 micrograms of E2 in 5 mL of 1:1 ethanol:saline or 5 mL of 1:1 ethanol:saline; 12 h later, ewes received i.v. injection of either 400 USP units of OT or saline. Jugular blood was collected on d 7, 8, 9, 10, 12, 14, 16, and 18. Progesterone concentrations were unaffected by the treatments. Experiment 2 was conducted to determine the dose of pFSH needed to induce approximately six corpora lutea (CL). Ten-day Norgestomet implants inserted between d 8-12 of the estrous cycle were used to synchronize estrus in Hampshire and Hampshire x Dorset ewes (n = 23). Ewes received a total of either 0, 18, 27, or 36 mg of pFSH, which was injected i.m. at -24, -12, 0, 12, 24, and 36 h relative to implant removal. The dose at each respective time was 19.4, 19.4, 16.7, 16.7, 13.9, and 13.9% of the total. Ewes received 400 IU of PMSG i.m. at -24 h. The CL were counted laparoscopically on d 6 (d 0 = estrus). Number of CL increased linearly (P < .01) with dose of pFSH; there were 1.8, 3.6, 6.3, and 11.2 CL/ewe, respectively. Experiment 3 was conducted to determine the effect of the E2-OT treatment, mode of transfer or the interaction of E2-OT treatment x mode of transfer on embryo survival and development. Experiment 3 was conducted over two breeding seasons and across two trials. In the first trial ewes were assigned to one of three randomized treatments. Procedural limitations that were later overcome prevented a true 2 x 2 factorial design; therefore, transcervical transfer without hormonal treatment was excluded in the first trial. In the second trial, ewes were assigned to a 2 x 2 factorial array of treatments. On d 6 of pregnancy, embryos rating a fair or better were transferred into recipients either transcervically or laparoscopically. Recipients were administered either an E2 (d 6) - OT (d 7) treatment or an ethanol:saline-saline treatment following the same protocol as in Exp. 1. Embryos were recovered on d 12 in Trial 1 and d 14 in Trial 2. Embryos were evaluated morphologically for development and ranked on a scale of one to four; one represented no development and four represented development to the morphological stages associated with the day of collection. The treatments did not affect the percentage of embryos recovered after transfer or the percentage of embryos that showed some developed. However, there was an effect of mode of transfer on mean rank of embryo development; embryos transferred laporscopically developed further than embryos transferred transcervically (P < .01). This may have been an artifact of a technician effect between trials. There was an effect of E2-OT treatment on transcervical transfer (P < .01), indicating that it may be detrimental to transfer embryos transcervically without dilating the cervix. In conclusion, the E2-OT treatment did not affect luteal function, and the E2-OT treatment can be used to dilate the cervix and enhance success of transcervical transfer of embryos. A 400 IU priming dose of PMSG and a total dose of 27 mg of pFSH can be used to induce the target number of six CL. / Master of Science

No description available.

Surgical embryo transfer in rodents is a common procedure in today’s research laboratory, although little is known of the effect analgesics may have on not only the recipient female but also the embryos. Two perioperative analgesics, ketoprofen and buprenorphine, were evaluated against a saline control in terms of number of pups born, number of pups weaned, and whether or not a litter was born. Both a uterine approach and an oviduct approach were evaluated. Post-surgical behavior was compared among the three surgical animals in each group, and between the non-surgical analgesic control and its surgical counterpart. Results indicated that ketoprofen and buprenorphine have no effect on the number of pups born, weaned, or litters born when compared to a saline control. Significant differences were found between the non-surgical analgesic control and its surgical counterpart in two behavioral categories; once for ketoprofen (behavior) and once for buprenorphine (physical condition). No other differences were found.

In vitro produced (IVP) bovine embryos are useful for investigating the mechanisms affecting early embryonic failure. The work in this thesis explored how interleukin-6 (IL6), an embryokine that increases inner cell mass (ICM) influences post-transfer embryonic survival and development of the embryo-proper and fetus. Four replicates of slaughterhouse-derived cumulus oocyte complexes underwent in vitro maturation and fertilization. On day 5 post-fertilization, embryos were treated with either 1% Bovine Serum Albumin (BSA) (CONT) or 100ng/mL recombinant bovine IL6 with 1% BSA (TRT). On day 7.5 post-fertilization, individual blastocysts were loaded into transfer straws. Beef and dairy cow recipients were synchronized with the day of in vitro fertilization using a 7-d CO-Synch protocol. A subset of cows from each group underwent fixed-time artificial insemination (AI) (n=37). The remaining cows underwent embryo transfer (ET) in the uterine horn ipsilateral to a corpus luteum (CL) (IL6 n=35; CONT n=51). Embryo and fetal measurements were performed via transrectal ultrasonography weekly from days 28-56 post-insemination, respectively. Overall pregnancy rates were 40.0% IL6; 19.6% CONT; and 32.4% AI. Crown-rump lengths (CRL) were reduced (P<0.05) in CONT pregnancies when compared with IL6 and AI at days 28, 35, 42, and 56. A tendency (P=0.057) for larger abdominal diameters was detected between IL6 and CONT groups. Also, IL6 had larger crown-nose lengths than CONT (P<0.05) and tended to be larger than AI (P=0.07). In summary, IL6 treatment produced pregnancies resembling AI-generated pregnancies more so than conventionally cultured embryos, supporting the hypothesis that IL6 improves developmental competency of IVP embryos. / Master of Science / The incidences of pregnancy loss in both beef and dairy cattle industries are profound and are upwards of 60 percent. Financial stability for both of these industries revolves closely around the ability of cattle to give birth to a live calf annually. While artificial insemination (AI) has been heavily adopted and utilized widely in the dairy industry, the use of in vitro produced (IVP) bovine embryos has shown promise in lessening some of the stresses placed on impregnating cattle. The IVP of bovine embryos serves as a strong model to understand how pregnancy losses occur. Briefly, IVP involves the collection of eggs from donor animals, and subsequent fertilization to mimic what occurs within the animal naturally. A disadvantage of in vitro produced embryos is their reduced likelihood to establish pregnancy after transfer into recipient animals. Interleukin-6 (IL6) was recently identified as a pro-developmental factor that may improve the quality and post-transfer competency of in vitro produced embryos. The objective of this work was to determine if IL6 supplementation during in vitro culture improves post-transfer fetal development. Oocytes (i.e. eggs) were retrieved from slaughterhouse-derived ovaries and subjected to in vitro maturation and fertilization. On day 5 post-fertilization, embryos were treated with either 0 (CONT; 1% BSA) or 100ng/mL recombinant bovine IL6. On day 7.5 post-fertilization, individual embryos (blastocyst stage) were loaded into transfer straws. Estrous synchronized beef (n = ) and dairy (n = ) cow recipients were allocated into treatment groups in the following manner. A subset of cows from each group underwent fixed-time AI (n=37). Remaining cows underwent embryo transfer (ET) in the uterine horn ipsilateral to a corpus luteum; 51 of these cows received a CONT embryo and the remaining 35 cows received an IL6 embryo. Thus, there were three treatment groups: AI, CONT, and IL6. Embryo and fetal measurements were performed via transrectal ultrasonography weekly from day 28 to 56, these included crown-rump length, crown-nose length, abdominal diameter, and amniotic vesicle. Pregnancies that remained throughout the entirety of the experiment 40.0% for IL6 (14/35); 19.6% for CONT (10/51); and 32.4% for AI (12/37). In summary, IL6 treatment of embryos produced pregnancies with characteristics more similar to the current industry standard of AI, rather than conventionally cultured embryos (CONT), supporting the hypothesis that IL6 supplementation to bovine embryos on day 5 post-fertilization improves developmental competency of in vitro produced embryos.

Superovulation of donor cows, embryo transfer, and estrus synchronization of recipients are widely used technologies in the purebred cattle industry. Progress continues to be made to achieve efficient and economic use of these technologies. The first retrospective study was conducted to compare embryo production between a stimulation protocol using only Folltropin as the gonadotropin, and a stimulation protocol using Folltropin and Pluset. Beefmaster donor cows (n=12) were stimulated using both protocols over two stimulated cycles, one protocol each cycle. Both protocols used the same synchronization protocol with only the gonadatropin injections differing. The control protocol (Folltropin protocol) consisted of seven Folltropin injections over the course of 3.5 days. The treatment protocol (Folltropin + Pluset protocol) consisted of four Folltropin injections followed by three Pluset injections over the course of 3.5 days. The mean numbers of viable embryos did not differ between treatments (P>0.01) and were 9.33 and 6.58 for the control and treatment protocols, respectively. The proportion of viable embryos to total ova for each protocol was 0.49 and 0.48 for the control and treatment protocols, respectively (P > 0.10). No significant difference on embryo production was observed between the control and treatment protocols. The second retrospective study was performed to compare pregnancy rates after embryo transfer between Beefmaster recipients who received eCG during synchronization and recipients who did not receive eCG during synchronization. Due to the conditions of this study, statistical analysis could not be performed. Pregnancy rates are reported, but they are not statistically significant. Recipients in the control group (n=332) were synchronized with a protocol using a CIDR insert for seven days, a progesterone estradiol injection at the time of CIDR insertion, a prostaglandin (PG) injection at CIDR removal, and an estradiol injection the day after CIDR removal. Recipients in the treatment group (n=142) were synchronized using the same synchronization protocol as the control group, except eCG was administered five days after CIDR insertion. Pregnancy rates were 44.88 and 38.73 for the control and treatment groups, respectively. The addition of eCG to the synchronization protocol did not appear to be either beneficial or detrimental to pregnancy rate under the conditions of this study. In summary, the addition of Pluset to the stimulation protocol for donors was not detrimental to embryo production. The estrus synchronization protocol with eCG for recipients did not appear to be beneficial; however, a controlled studies are still warranted to further investigate the potential effects of recipient age, parity, body condition score, or breed effect on response to eCG.

A series of experiments were designed to study the effects of a purified equine pituitary extract product containing a high FSH to LH ratio (eFSH) on superovulation and reproductive performance in mares. A significance level of P < 0.05 was used for the data analyses.<p> The treatment protocol included twice daily administration of 12.5 mg eFSH beginning at a follicular diameter of ¡Ý20 or 25 mm. The treatment was stopped when a preovulatory-sized follicle was detected (¡Ý35mm), and subsequently human chorionic gonadotropin (hCG) was administered to induce ovulation(s). The eFSH treatment significantly stimulated the ovaries of cycling and vernal transitional mares. This resulted in the development of multiple preovulatory-sized follicles, increased the number of ovulations, and enhanced donor embryo recovery rates. In mares which ovulated, approximately 70% of embryo recovery attempts resulted in the recovery of ¡Ý1 embryo. However, incidences of ovulation failure and non-ovulatory follicles were significantly higher compared to control mares. Furthermore, there were significant variations in the superovulatory response to eFSH among cycling and vernal transitional mares in the same study, and among different studies, in terms of number of ovulations, number of embryos and embryo per ovulation rates.<p> Administration of eFSH significantly modified reproductive tract variables (tone and edema) and serum concentrations of progesterone (P4) and estradiol-17¦Â (E2) on the days that oocyte maturation, fertilization, and early embryonic development were expected to occur. The administration of eFSH was also significantly associated with lower quality scores in a proportion of embryos recovered, and lower than expected pregnancy rates in recipients which received an embryo recovered from eFSH-treated cycling donor mares as compared to embryos from non-stimulated control mares. Moreover, eFSH treatment did not significantly increase pregnancy rate per estrous cycle in mares intended to carry their own pregnancy; however, the incidence of twin pregnancy tended to increase.<p> The effects of estrus synchronization regimens employed prior to eFSH treatment initiation were examined in cycling mares. A progesterone and estradiol regimen (P&E) was significantly more efficient than PGF2¦Á administration in diestrus for ovulation synchrony among eFSH-treated mares, with ¡Ý80% of mares ovulating within a 3 day period. The superovulatory outcomes (proportion of mares that ovulated, number of ovulations and embryo recovery), however, were significantly lower than those obtained with PGF2¦Á administration.<p> In vernal transitional mares, eFSH treatment resulted in a significantly higher number of preovulatory-sized follicles and a greater number of ovulations, compared to vernal transitional mares treated with deslorelin or porcine-FSH, or as compared to control mares. Most transitional mares (73% to 100%) ovulated after a mean of 5 days of eFSH treatment. These ovulations resulted in pregnancies and/or successful embryo recoveries. Following eFSH treatment in vernal transition, the first inter-ovulatory interval of the breeding season was significantly prolonged (>21 d) in about half of the mares.<p> In summary, eFSH treatment significantly stimulated follicular growth and multiple ovulations in cycling mares and in vernal transitional mares. The treatment significantly increased reproductive efficiency of cycling mares in terms of embryo recovery rates, and in vernal transitional mares in terms of establishing pregnancies or recovering embryos early in the breeding season. However, the eFSH treatment significantly altered the hormonal environment (E2 and P4), and was associated with modifications in follicular growth, ovulation, and embryo parameters. These aspects should be considered in the development of superovulation protocols for mares in future studies.

A series of experiments were designed to study the effects of a purified equine pituitary extract product containing a high FSH to LH ratio (eFSH) on superovulation and reproductive performance in mares. A significance level of P < 0.05 was used for the data analyses.<p> The treatment protocol included twice daily administration of 12.5 mg eFSH beginning at a follicular diameter of ¡Ý20 or 25 mm. The treatment was stopped when a preovulatory-sized follicle was detected (¡Ý35mm), and subsequently human chorionic gonadotropin (hCG) was administered to induce ovulation(s). The eFSH treatment significantly stimulated the ovaries of cycling and vernal transitional mares. This resulted in the development of multiple preovulatory-sized follicles, increased the number of ovulations, and enhanced donor embryo recovery rates. In mares which ovulated, approximately 70% of embryo recovery attempts resulted in the recovery of ¡Ý1 embryo. However, incidences of ovulation failure and non-ovulatory follicles were significantly higher compared to control mares. Furthermore, there were significant variations in the superovulatory response to eFSH among cycling and vernal transitional mares in the same study, and among different studies, in terms of number of ovulations, number of embryos and embryo per ovulation rates.<p> Administration of eFSH significantly modified reproductive tract variables (tone and edema) and serum concentrations of progesterone (P4) and estradiol-17¦Â (E2) on the days that oocyte maturation, fertilization, and early embryonic development were expected to occur. The administration of eFSH was also significantly associated with lower quality scores in a proportion of embryos recovered, and lower than expected pregnancy rates in recipients which received an embryo recovered from eFSH-treated cycling donor mares as compared to embryos from non-stimulated control mares. Moreover, eFSH treatment did not significantly increase pregnancy rate per estrous cycle in mares intended to carry their own pregnancy; however, the incidence of twin pregnancy tended to increase.<p> The effects of estrus synchronization regimens employed prior to eFSH treatment initiation were examined in cycling mares. A progesterone and estradiol regimen (P&E) was significantly more efficient than PGF2¦Á administration in diestrus for ovulation synchrony among eFSH-treated mares, with ¡Ý80% of mares ovulating within a 3 day period. The superovulatory outcomes (proportion of mares that ovulated, number of ovulations and embryo recovery), however, were significantly lower than those obtained with PGF2¦Á administration.<p> In vernal transitional mares, eFSH treatment resulted in a significantly higher number of preovulatory-sized follicles and a greater number of ovulations, compared to vernal transitional mares treated with deslorelin or porcine-FSH, or as compared to control mares. Most transitional mares (73% to 100%) ovulated after a mean of 5 days of eFSH treatment. These ovulations resulted in pregnancies and/or successful embryo recoveries. Following eFSH treatment in vernal transition, the first inter-ovulatory interval of the breeding season was significantly prolonged (>21 d) in about half of the mares.<p> In summary, eFSH treatment significantly stimulated follicular growth and multiple ovulations in cycling mares and in vernal transitional mares. The treatment significantly increased reproductive efficiency of cycling mares in terms of embryo recovery rates, and in vernal transitional mares in terms of establishing pregnancies or recovering embryos early in the breeding season. However, the eFSH treatment significantly altered the hormonal environment (E2 and P4), and was associated with modifications in follicular growth, ovulation, and embryo parameters. These aspects should be considered in the development of superovulation protocols for mares in future studies.

Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-12-11Bitstream added on 2014-06-13T18:07:44Z : No. of bitstreams: 1 alonso_ma_me_botfmvz.pdf: 646083 bytes, checksum: cd0a270ab9839fdf6c856789eacab1c4 (MD5) / Universidade Estadual Paulista (UNESP) / A técnica de transferência de embrião (TE) foi desenvolvida nos anos 70, e vem sendo cada vez mais difundida. A eficiência de um programa de TE será determinada pelos índices de recuperação embrionária e taxa de prenhez. A receptora de embrião consiste no ponto crítico do programa de TE, e a correta seleção deste elo (fundamental) da cadeia é imprescindível para o sucesso da técnica. O objetivo deste trabalho foi avaliar o efeito do dia do ciclo da receptora sobre as taxas de prenhez pós transferência de embrião; o efeito do tônus e morfoecogenicidade uterina nas taxas de prenhez de receptoras de embrião; relacionar a concentração plasmática de progesterona em éguas candidatas à receptora de embrião classificadas como marginais e aceitáveis e relacionar as concentrações plasmáticas de progesterona em éguas candidatas à receptora de embrião apresentando diferentes tônus e morfoecogenicidade uterina. Éguas receptoras entre os dias 3 a 8 pós ovulação foram utilizadas. A morfoecogenicidade e tônus uterino no dia da TE foram avaliados. O diagnóstico de prenhez foi realizado no dia 15 do embrião. Utilizou-se o Qui-Quadrado para análise das taxas de prenhez nos dias do ciclo, assim como para as taxas de prenhez e características uterinas. Para avaliação do efeito da concentração de progesterona sobre a classificação foi utilizada ANOVA. A comparação das concentrações plasmáticas de progesterona com as características uterinas foi feita utilizando-se o teste t. As taxas de prenhez não foram afetadas pelo dia do ciclo da receptora. As éguas apresentando morfoecogenicidade 1 e 2 tiveram maiores taxas de prenhez do que as éguas com morfoecogenicidade 3, diferindo estatisticamente (p<0,05). As éguas com morfoecogenicidade uterina 4 apresentaram índices de prenhez inferiores estatisticamente aos 3 outros padrões. / Embryo transfer (ET) technique was developed in the seventies and nowadays it is spreading consistently. The efficiency on an ET programme is determined by recovery rate and pregnancy rate. The recipient mare is the most important factor in the ET programme, with special emphasis on its selection. The aim of this study was to evaluate the effect of the recipient’s day of cycle on pregnancy rate after transfer, the effect of uterine tone and morphoecogenicity in pregnancy rates, relate plasmatic progesterone concentration in candidate recipient mares that were qualified as acceptable or marginal, and to relate plasmatic progesterone concentration to different uterine tone and morphoecogenicity in candidate recipient mares. Recipient mares were used between day 3 and 8 post ovulation. Morphoecogenicity and uterine tone were evaluated on ET day. Pregnancy diagnosis was performed on day 15 of the embryo. Chi square test was used to analyze pregnancy rates on the different days of cycle, and to relate pregnancy rates and uterine characteristics. In order to evaluate progesterone concentration in recipients that were classified as marginal or acceptable, ANOVA was utilized. To compare progesterone concentration and uterine characteristics, t test was performed. Pregnancy rates were not influenced by recipient’s day of cycle on ET day. Mares presenting morphoecogenicities 1 and 2 had higher pregnancy rates than mares presenting morphoecogenicity 3, being statistically different (p<0,05). Mares presenting morphoecogenicity 4 had the lowest pregnancy rate, compared to the others. Uterine tone affected pregnancy rate. Mares with uterine tone 1 had higher pregnancy rates than uterine tone 2 and 3. Mares presenting uterine tone 2 had lower pregnancy rate than uterine tone 1, but higher than uterine tone 3 (p<0,05). Average plasmatic progesterone concentration on day 8 post xvi ovulation was higher than on day 4 post ovulation, with p<0,0002.

The objectives of this study were to evaluate the effects of flunixin meglumine (FM), an inhibitor of PGF2a synthesis, administered at the time of embryo transfer (ET) and insertion of an intravaginal progesterone-releasing device (CIDR) at the time of ET on pregnancy rates (PR) and the resynchronization of estrus. Beef cows (n = 796) and heifers (n = 108) at three locations were assigned randomly within age to one of four groups in a 2 x 2 factorial arrangement of treatments with injection of FM (500 mg i.m.; Phoenix Scientific, St. Joseph, MO) 2 to 12 min prior to ET and insertion of a CIDR (1.38 g progesterone; Pfizer, New York, NY) for 13 d immediately following ET as main effects. Fresh or frozen embryos (Stage = 4 or 5; Grade = 1 or 2) were randomly assigned to be transferred to recipients on d 6 to 9 of the estrous cycle. At Location 2 a subset of fresh embryos were split and transferred as fresh half embryos (n = 192). Recipients at Location 2 only (n = 493) were observed for signs of return to estrus beginning 9 d after ET. Recipients that returned to estrus at Location 2 were either bred by AI 12 h after estrus or received an embryo 7 d after estrus. Pregnancy was diagnosed by ultrasonography. Pregnancy rates were analyzed using the LOGISTIC procedure of SAS. Pregnancy rates of split embryo recipients were analyzed separately using the same statistical procedure. Variation in the timing of the return to estrus was determined by an F-test for heterogeneity of variances. Following the initial ET, pregnancy rates of recipients receiving whole embryos were not affected by CIDR administration (P > 0.05; 65% with CIDR, 70% without CIDR), however, there was a significant FM x location interaction on PR (Location 1, 89 vs. 57%; Location 2, 69 vs. 64%; Location 3, 64 vs. 67% for FM vs. no FM, respectively). There was a significant CIDR x FM interaction on PR of recipients receiving split embryos. Pregnancy rates of split embryo recipients receiving CIDR treatment (54%) or FM treatment alone (41%) were less than controls (64%). However, recipients receiving both CIDR and FM had PR similar to controls (60%). The timing of the return to estrus was more synchronous (P < 0.01) for recipients fitted with a CIDR, but PR of recipients bred following a return to estrus did not differ in cows receiving or not receiving a CIDR (P > 0.13; 68 and 62%, respectively). Effects of FM on PR were location dependent and CIDR insertion at ET improved synchrony of the return to estrus. / Master of Science