Porcine Follicle-stimulating Hormone Research Articles

The pFSH dose affects the efficiency of in vivo embryo production in Santa Inês ewes

This study compared the follicular growth, superovulatory response, and in vivo embryo production after administering two doses of porcine follicle-stimulating hormone (pFSH) in Santa Inês ewes. The estrous cycle of 36 multiparous ewes was synchronized with the Day 0 protocol and superovulated with 133 mg (G133, n=18) or 200 mg (G200, n=18) of pFSH. Ultrasonographic evaluations of the ovaries were performed, ewes were mated and submitted to non-surgical embryo recovery. Viable blastocysts were stained with Nile Red and Hoechst. The G200 had a greater number of medium and large follicles, as well as a larger size of the third largest follicle. A total of 97.2% (35/36) of the ewes came into estrus and it was possible to transpose cervix in 80.6% (29/36). There were no effects of treatments in the response to superovulation, the proportion of ewes in which was possible to transpose the cervix, the number of corpora lutea, the number of anovulatory follicles, the proportion of ewes flushed with at least one recovered structure, number of recovered structures, number of viable embryos, viability rate, and recovery rate. The G200 ewes were in estrus for a longer period of time than the G133 ewes (54.0 ± 4.5 h vs. 40.3 ± 3.6 h) and produced more freezable embryos (6.5 ± 1.6 vs. 2.3 ± 0.7) than G133. Both doses promoted an efficient superovulatory response and did not affect embryonic lipid accumulation. The dose of 200 mg of pFSH showed greater potential to increase the superovulatory response, as it increased follicular recruitment and the recovery of freezable embryos.

Evaluation of a long-acting recombinant bovine FSH for multiple ovulation and embryo transfer in dromedary camels

The present study was conducted to test a new super-agonist recombinant bovine FSH (rbFSH) to induce superovulation (SOV) in dromedary camels. In experiment I, a single IM injection of 40, 60, 80, 100, or 120 µg rbFSH was administered (4 donors per group) to determine the effective dose resulting in acceptable multiple ovulation and embryo yield. Administration of 40 µg was ineffective, while 100 and 120 µg were associated with increased numbers of developed follicles, corpora lutea, and recovered embryos compared to administration of 60 and 80 µg. In experiment II, donors were divided into treatment groups to compare rbFSH with two conventional protocols for SOV. Donors received a single dose of 2000 IU eCG in combination with 400 mg porcine follicle-stimulating hormone (pFSH; Folltropin-V®; Group 1, n = 29) or 500 µg of pFSH with 100 µg of pLH (Stimufol®; Group 2, n = 16). Group 3 (n = 19) received a single dose of 100 µg rbFSH. No difference was found in the size and number of follicles per donor. Response time, ovulation rate, and the number of corpora lutea and recovered embryos per donor were similar in all groups. The number of medium-sized and transparent embryos decreased while the number of small-sized and semi-transparent embryos increased in Group 3 (rbFSH) compared to the other two groups. The pregnancy rate of the recipients at 10 days post-ET, at two months of gestation, and the rate of early pregnancy loss (EPL) did not differ among the groups. In conclusion, a single IM administration of 100 µg rbFSH induces a successful superovulation in dromedary camels and has the advantage of reducing stress associated with multiple FSH administration of the conventional protocols.

190 Assessment of porcine follicle-stimulating hormone delivery mode before ovum pickup and in vitro embryo production in pregnant heifers

190 Assessment of porcine follicle-stimulating hormone delivery mode before ovum pickup and in vitro embryo production in pregnant heifers

Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein.

Pichia pastoris is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that N-acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHβ secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing β-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHβ production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHβ. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHβ secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC.

Luteal Function, Biometrics, and Echotextural Attributes in Santa Inês Ewes Superovulated with Different Total Doses of Porcine Follicle-Stimulating Hormone

Premature regression of corpora lutea (PRCL) may adversely affect the outcome of hormonal ovarian superstimulation in small ruminants, and the total dose of exogenous gonadotropins used may be one of the causes of this condition. There were two major objectives of the present study: (1) to evaluate the effects of different superovulatory doses of porcine follicle-stimulating hormone (pFSH) on the biometry, blood perfusion (Doppler), and echotextural characteristics of luteal structures; and, (2) to determine the usefulness of biometric, vascular, and echotextural luteal variables, as well as measurements of circulating progesterone (P4) concentrations for early detection of PRCL in superovulated Santa Inês ewes. Twenty-seven Santa Inês ewes received an intravaginal P4-releasing device (CIDR) from Days 0 to 8 (Day 0 = random day of the anovulatory period). An IM injection of d-cloprostenol (37.5 μg) was given at the time of the CIDR insertion and withdrawal. On Day 6, all the ewes received 300 IU of eCG IM and were divided into three treatment groups (each n = 9): G100 (100 mg); G133 (133 mg); and G200 (200 mg of pFSH) administered IM every 12 h in eight injections. Transrectal ovarian ultrasonography and jugular blood sampling for serum P4 measurements were performed on Days 11 to 15. On the day of embryo recovery (Day 15), all the ewes underwent diagnostic videolaparoscopy and were classified, based on their luteal characteristics, into three response groups: nCL (ewes with normal CL only); rCL (ewes with regressing CL only); and ewes with both nCL and rCL following the superovulatory regimen. Our present results indicate that the total pFSH doses of 100 mg and 200 mg result in similar ovulatory responses and luteal function/biometrics, although the percentage of donor ewes with nCL was greater (p < 0.05) for G100 compared with the G200 animals. An application of 133 mg of pFSH was associated with diminished luteogenesis. Lastly, circulating P4 concentrations, ultrasonographic estimates of total luteal area, and CL pixel heterogeneity (standard deviation of numerical pixel values) are promising markers of luteal inadequacy in superovulated ewes.

Haematological Responses following Oestrus synchronisation and superovulation protocols in West African Dwarf Goats

Experiments were performed to evaluate the haematological (erythrocyte and leucocyte parameters) response of West African Dwarf goats following oestrus synchronisation and superovulation protocol. Twenty-four clinically healthy female goats aged 2.34 ± 0.4 years and average body weight of 13.83 ± 0.7 kg was used for the experiment. The goats were divided into two groups. Group I goats was synchronized using Controlled Internal Drug Release (CIDR) for 18 days with the first day of insertion as day 0. On day 16 following CIDR insertion into the vagina, a single dose of 500 IU equine chorionic gonadotropin (eCG) was administered to each doe intramuscularly while group II served as the control was subjected to the same housing and environmental conditions but was not synchronized. superovulation was induced with porcine follicle stimulating hormone (pFSH) which was injected intramuscularly at a rate of 5mg/kg in reducing doses twice a day at 06.00 h and 18.00 h for 3 days starting from 48hr i.e., 2 days before CIDR removal (day 17). A blood sample of 5 mL was collected by jugular venipuncture of each of the goats before the protocol (Day 0) and at the end of the protocol (Day 18). All erythrocyte parameters evaluated showed a significantly higher value in group II (control) compared to group I post-oestrus synchronisation while total leucocyte count, neutrophil count and neutrophil/lymphocyte ratio values obtained after the protocol were also higher in group I (P &lt; 0.05) compared to group II. It is therefore concluded that oestrus synchronisation and superovulation may elicit stress in West African Dwarf goats and an appropriate antioxidant should be part of the protocol.

Enhancement of HSA-pFSHβ production by disrupting YPS1 and supplementing N-acetyl-L-cysteine in Pichia pastoris.

Pichia pastoris is widely used for the production of recombinant proteins, but the low production efficiency hinders its wide application in biopharmaceuticals. Moreover, many biopharmaceutical-like proteins are accompanied by degradation during secretory expression in P. pastoris. In this study, we used human serum albumin and porcine follicle-stimulating hormone β (HSA-pFSHβ) fusion protein as a model protein to investigate whether YPS1 and YPT7 gene disruption and N-acetyl-L-cysteine (NAC) supplementation have synergistic effects to inhibit the degradation of recombinant proteins. Our results showed that YPS1 gene disruption reduced the degradation of intact HSA-pFSHβ and increased the yield of intact protein in the culture medium and cells without affecting the integrity of the cell wall. Moreover, the beneficial effects of YPS1 gene disruption were associated with the upregulation of the MAPK signaling pathway and maintenance of redox homeostasis. YPS1 gene disruption and NAC supplementation had synergistic effects on HSA-pFSHβ production. In addition, disruption of vacuolar morphology by YPT7 gene disruption or NH4Cl treatment affected the production of recombinant HSA-pFSHβ protein. Furthermore, YPT7 gene disruption inhibited the processing of signal peptide in high-level produced HSA-pFSHβ strain. In conclusion, our results demonstrated that YPS1 disruption could reduce the degradation of intact HSA-pFSHβ proteins, and synergistically increase the yield of intact HSA-pFSHβ with NAC supplementation. This study provided a valuable reference for reducing recombinant protein degradation and therefore improving the yield of recombinant proteins in P. pastoris.

246 Luteal function, biometrics, and echotextural attributes in Santa Ines ewes superovulated with three different doses of porcine follicle stimulating hormone

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Serum anti-Müllerian hormone concentrations in female alpacas: variations during the reproductive cycle and correlation with ovarian superstimulation response

Earlier, we validated an anti-Müllerian hormone (AMH) enzyme-linked immunosorbent assay kit for male alpacas. First, wecompared the validation data with another kit. There was a high correlation (R2 = 0.94) between these 2 kits. Second, we used thelatter kit to determine serum AMH concentrations during follicular and luteal phases of the reproductive cycle in female alpacas.There were no differences (p = 0.39) in serum AMH concentrations in alpacas (n = 11) between peak follicular and luteal phases(mean ± SEM, 1.33 ± 0.35 versus 1.18 ± 0.34 ng/ml, respectively). Third, we treated female alpacas (n = 13; 5 - 11 years) after 14-day treatment with decreasing doses of porcine follicle-stimulating hormone. There was no effect (p &gt; 0.05) of day of treatmenton serum AMH concentrations. Number of follicles (7 - 10 mm; mean ± SD [as determined via transrectal ultrasonography]) atend of treatment (12.69 ± 5.25; range: 6 - 24) was positively correlated (R2 = 0.7; p &lt; 0.01) with serum AMH concentrations. Toconclude, the kit tested is usable for female alpacas; serum AMH concentrations were not affected by the cycle stage nor by ovariansuperstimulation treatment. Furthermore, a significant correlation between serum AMH serum concentrations and response to superstimulationsuggested that estimation of serum AMH concentrations may be valuable in determining ovarian follicular reserve.

Synchronization of follicle wave emergence before ovarian superstimulation with FSH and ovum pick-up improves in vitro embryo production in pregnant heifers

The objective of the present study was to evaluate effects of synchronization of timing of follicle wave emergence, before ovarian superstimulation and ovum pick-up (OPU), on ovarian response and embryo production in pregnant heifers. Pregnant (47–69 days of gestation) Holstein heifers (n = 64), 19.0 ± 0.3 months of age, were assigned in a completely randomized design to one of two groups: synchronization of follicular wave emergence using follicle ablation (Synchronized) or untreated control (Non-synchronized). Superstimulatory treatments consisting of 160 mg (280 IU) of porcine follicle stimulating hormone (p-FSH), administered in four decreasing dose treatments 12 h apart, were initiated 36 h after follicle ablation or at random stages of the follicular wave in heifers of the Synchronized and Non-synchronized group, respectively. Ovum pick-up was performed in all heifers 40 h after the last p-FSH administration and retrieved cumulus oocyte complexes (COCs) were subjected to in vitro embryo production (IVEP) procedures. Ultrasonography was performed immediately before OPU to determine number and size of ovarian follicles. Differences in treatment responses between groups were evaluated using generalized linear mixed models. Total number of follicles at the time of OPU was not different between treatment groups (P = 0.61), however, the number of small follicles (<6 mm) was greater (P = 0.05) in heifers of the Non-synchronized group, whereas number of medium size follicles (6–10 mm) tended (P = 0.09) to be greater in heifers of the Synchronized group. Heifers in the Synchronized group had a greater (P = 0.01) percentage of medium sized follicles and a lesser (P = 0.01) percentage of smaller sized follicles than heifers in the Non-synchronized group. There were no differences (P > 0.15) in total number of recovered COCs, or number of viable COCs between groups. Cleavage percentage (84.5% and 72.8%) and blastocyst percentage (48.2% and 33.4%) were greater (P < 0.01) in heifers of the Synchronized than Non-synchronized group, respectively. As a result, mean number of blastocysts per OPU/heifer was greater (P = 0.006) in the heifers of the Synchronized (8.9 ± 1.0) than the Non-synchronized (5.5 ± 0.9) group. In conclusion, synchronizing the time of follicle wave emergence in pregnant heifers, prior to ovarian superstimulation with FSH and OPU results in a greater superstimulatory response and oocyte competence leading to greater embryo production.

Comparison of superovulatory responses to a standardized hormonal superstimulation protocol among three indigenous breeds of sheep in Brazil

This study examined the differences in ovarian responses and in vivo embryo production in three naturalized Brazilian sheep breeds (Morada Nova, Santa Inês, and Somalis Brasileira). Thirty cycling, multiparous ewes (n = 10/breed) received intravaginal devices containing 0.33 g of progesterone (CIDR®) for 9 days and the twice-daily superovulatory treatment with 133 mg of porcine follicle-stimulating hormone (six decreasing doses) started on Day 7 (Day 0 PM = CIDR® insertion). On Days 11 (36 h after CIDR® removal) and 15 (12 h before non-surgical embryo recovery performed seven days after CIDR® removal), all ewes were examined using B-mode and Doppler ultrasonography. The number of medium-sized ovarian follicles (4.0–5.99 mm) on Day 11 was greater (P = 0.003) in Santa Inês and Somalis Brasileira compared with Morada Nova ewes. Small antral follicle numbers (2.0–3.99 mm in diameter) on Day 11 were directly related to ovulatory responses in all three breeds of superovulated ewes. The number of corpora lutea on Day 15 was greater (P < 0.0001) in Santa Inês, followed by Somalis Brasileira and Morada Nova ewes; however, the number of viable embryos and viability rate did not differ (P > 0.05) among the three genotypes of ewes studied. The total ovarian area (TA), color Doppler area (DA), and DA/TA × 100% increased from Day 11 to Day 15 in all breeds, and they were lowest (P < 0.001) in superovulated Morada Nova sheep. Significant positive correlations among ovarian antral follicle numbers in different size classes (Day 11) and ovulatory responses were recorded in all breeds, but associations between the ovarian blood perfusion and superovulatory outcomes were restricted to the Morada Nova and Santa Inês genotypes. In summary, ovarian follicle numbers and blood flow indices differed among the three naturalized Brazilian ewes, but no significant differences were noted in embryo yields and quality following superovulation. Small antral follicle count 36 h after CIDR® treatment was a reliable predictor of impending ovulation rates in all ewes under study.

Study of the factors affecting embryo yields and quality in superovulated Morada Nova ewes that underwent non-surgical uterine flushing.

The present study compared the outcomes of in vivo embryo production in Morada Nova ewes subjected to either 9-day (G-9SOV , n=21) or 12-day (G-12SOV , n=21) progesterone (P4 )-based estruses synchronization protocol coupled with superovulatory treatment with decreasing doses of porcine follicle-stimulating hormone (133mg of pFSH given over 3days). Non-surgical embryo recovery (NSER) was performed 6-7days after the onset of oestrus. Total antral follicle count doubled from the first to the sixth pFSH dose in both groups (p<.05). Oestrus responses did not vary between the two groups of animals (95.2%). Corpora lutea (CL) were detected in 85.0% and 60.0% of ewes that previously manifested oestrus behaviour in G-9SOV and G-12SOV respectively. NSER was successfully completed in 86.2% of ewes that had CL (p>.05). The mean number of CL per ewe/successfully flushed donor ewe was greater (p<.05) in G-12SOV (12.3±1.7/12.1±1.9) than in G-9SOV (7.9±1.4/8.2±1.6). Mean numbers of retrieved blastocysts and viable embryos were greater (p>.05) in G-12SOV (5.8±1.9 and 3.7±1.7) than G-9SOV (3.5±1.1 and 0.8±0.3 respectively). The total follicle count (all follicles ≥2mm in diameter) at the sixth pFSH dose (at P4 -device removal) was positively correlated (p<.05) with the number of CL (r=.95) and viable embryos (r=.91) in G-12SOV . The ewes with ≥10 Cl (48% of all flushed donors) yielded 80.5% of viable embryos. In summary: (a) Morada Nova ewes from G-12SOV group had better superovulatory responses compared with G-9SOV group; (b) total follicle count at the last pFSH dose was a good predictor of superovulatory responses only in the ewes primed with P4 for 12days; and (c) animals with ≥10 ovulations are main contributors to viable embryo production in Morada Nova ewes.

Plasma profile of follicle-stimulating hormone and sex steroid hormones after a single epidural administration of follicle-stimulating hormone via caudal vertebrae in Holstein dry cows.

The conventional follicle-stimulating hormone (FSH) treatment for bovine superstimulation involves multiple intramuscular injections, which is stressful for animals and onerous. We herein investigated whether a single epidural injection of porcine FSH (pFSH) can induce superovulation and peripheral concentrations of pFSH and steroid hormones after the treatment in Holstein dry cows. We intramuscularly administered pFSH twice daily to three cows for 3 days (control) or a single epidural pFSH administration (epidural). Numbers of follicles (≥10mm in diameter) at estrus and corpora lutea at luteal phase were counted by ultrasonography. Blood was sampled from 0 to 104 h after the first pFSH administration and plasma pFSH, progesterone, androstenedione, testosterone, and estradiol-17β concentrations were measured. Numbers of follicles (control: 18.3 ± 7.5, epidural: 15.7 ± 4.0; mean ± SD) and corpora lutea (control: 7.3 ± 4.2, epidural: 8.0 ± 2.6) were similar between both treatments. Plasma pFSH concentrations were higher in epidural than in control (p < 0.01). Although no significant differences were observed in progesterone, androstenedione, or estradiol-17β concentrations between the groups, testosterone concentrations were slightly lower with the epidural treatment than with the control treatment (p = 0.08). In conclusion, superovulation was induced by a single epidural injection of pFSH, which achieved higher pFSH level than the multiple injections in Holstein dry cows.

Nonsurgical Embryo Recovery as a Feasible Tool for Supporting Embryo Biobanks of Locally Adapted Brazilian Sheep and Goats.

This study assessed the outcomes of nonsurgical embryo recovery (NSER) after superovulation (SOV) in five locally adapted Brazilian breeds of sheep and goats. The objective was to evaluate the feasibility and efficiency of using SOV combined with a less-invasive embryo collection technique for supplying the Brazilian animal gene bank with germplasm from specific genotypes of interest. Morada Nova (n = 20), Santa Inês (n = 20), and Somalis (n = 20) ewes received an intravaginal progesterone (330 mg) device for 9 days, while Canindé (n = 15) and Moxotó (n = 15) goats received an intravaginal medroxyprogesterone acetate (60 mg) device for 6 days. All females received 133 mg of porcine follicle-stimulating hormone (pFSH) administrated in six decreasing doses 12 hours apart, starting 60 hours before device removal, plus 37.5 μg of d-cloprostenol at the fifth and sixth pFSH dose. Donors in estrus were mated with fertile males. The corpora lutea (CL) number was assessed by ultrasonography 1 day before NSER. On day 6.5 or 7 after estrus, NSER was performed following hormonally induced cervical relaxation. A total of 97% of sheep and 90% of goats responded with estrus, and among those, 91% of sheep and 85% of goats presented a CL. In ewes, the numbers of CL were greater (p < 0.05) in the Santa Inês breed, while similar (p > 0.05) CL numbers were found among the goat breeds. All viable embryos were freezable (excellent and good quality) and the number per donor was 7.8 for sheep and 4.9 for goats. All parameters of NSER efficiency, embryo yield, and fertility post-NSER did not differ (p > 0.05) between breeds among each species. The SOV-NSER procedures applied for an embryo biobank supply of locally adapted Brazilian breeds of small ruminants were efficient regarding production of cryopreservable embryos, and preservation of donor fertility. Therefore, SOV followed by NSER is recommended for embryo biobank assembly in sheep and goats.

The mechanism of N-acetyl-l-cysteine in improving the secretion of porcine follicle-stimulating hormone in Pichia pastoris.

Our previous study revealed that N-acetyl-l-cysteine (NAC) could enhance the secretion of recombinant proteins by Pichia pastoris, but the corresponding molecular mechanisms are still unclear. In the present study, we explored whether other thiols have a similar action on the secretion of recombinant human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ), to reveal the mechanism of NAC on HSA-pFSHβ secretion. Transcriptome analysis showed that genes involved in oxidoreductase activity and oxidation-reduction process were upregulated in cells supplemented with NAC. The other three thiol-reducing regents including dimercaptopropanol (DT), thioglycolic acid, and mercaptolactic acid could improve HSA-pFSHβ production in the culture supernatant. Among them, only DT had similar effect as NAC on HSA-pFSHβ secretion and the increase of GSH content. Moreover, 1-20 mM GSH, 1-10mM cysteine, or 1-20 mM N-acetyl-d-cysteine supplementation could improve the secretion of HSA-pFSHβ. Furthermore, 0.4-3.2mM ethacrynic acid, rather than 1-16 mM BSO could inhibit the effect of NAC on the production of HSA-pFSHβ. These results indicated that NAC improved the secretion of HSA-pFSHβ by increasing the intracellular GSH content through its thiol activity rather than as a precursor for GSH synthesis. In conclusion, our results demonstrate, for the first time, that the secretion of recombinant HSA-pFSHβ in Pichia pastoris could be improved through thiol-reducing agent supplementation, and the mechanism of the effect NAC has on HSA-pFSHβ secretion is associated with improving the intracellular GSH content.

Comparison of cattle embryo collection between Ringer’s lactate solution and Dulbecco’s phosphate-buffered saline

ABSTRACT The objective of this study was to determine the efficiency of Ringer’s lactate solution (RL) and RL + 1% fetal bovine serum (FBS) and compare them with the efficiency of Dulbecco’s phosphate-buffered saline (D-PBS). Twenty-two Wagyu female cattle were subjected to superovulation and were randomly distributed to form three groups: group 1 – uterine flushing with RL (n = 8), group 2 – uterine flushing with RL + 1% FBS (n = 7), and group 3 – uterine flushing with D-PBS (n = 7, control group). Cows received a CIDR® device containing 1.9 g of progesterone at random stages of the estrous cycle (day 0). Progesterone withdrawal occurred on day 8 in the morning. For heifers, 160 mg of porcine follicle-stimulating hormone (FSH-P) was used and for cows, 200 mg. Prostaglandin F2α was also injected on the eighth day of FSH-P administration. On day 9, in the morning, hCG was administered. Females were superovulated and inseminated twice in a fixed time for embryo transfer. On the 16th day, females were subjected to uterine flushing for embryo collection. We collected 76 embryos from 22 females subjected to superovulation, of which 52 were transferable and 24 had degenerated. The total of embryos collected was 23, 16, and 23 for groups 1, 2, and 3, respectively. The embryo recovery rates per group were 13.86±4.23, 15.39±4.61 and 27.16±13.33%, in groups 1, 2, and 3, respectively. The means for the total structures collected per female were 2.88±0.85, 3.00±1.23, and 4.57±1.72 in groups flushed with RL, RL + 1% FBS, and D-PBS, respectively. We conclude that Ringer’s lactate solution and Ringer’s lactate solution + 1% of FBS and Dulbecco’s phosphate-buffered saline showed no significant differences in terms of embryo quality or quantity, suggesting that Ringer’s lactate solution is an alternative for collecting embryos in cattle.

NonSurgical Embryo Recovery from Estrus-Synchronized or Superovulated Morada Nova Ewes: A Feasible Strategy for Sheep Embryo Banking.

This study assessed the feasibility of in vivo embryo production and nonsurgical embryo recovery (NSER) in Morada Nova ewes (an endangered native Brazilian breed of sheep) subjected to different estrus synchronization and/or superovulation protocols. Ewes received intravaginal sponges soaked with 60 mg medroxyprogesterone acetate (MAP), which were kept in place for six (G6; n = 12), nine (G9; n = 12), or 12 (G12; n = 12) days. Half of the ewes in each group remained estrus synchronized only (SYNCH) and the other half was superovulated (SOV) with 133 mg porcine follicle-stimulating hormone (pFSH). There were no differences (p > 0.05) in antral follicle counts determined with ultrasonography 60 hours before MAP sponge removal (or at the time of the first pFSH dose) among G6 (6.4 ± 0.9), G9 (6.2 ± 0.7), and G12 (5.5 ± 0.6). Estrus responses and NSER success rates did not vary (p > 0.05) among the three progestin-treatment groups of ewes for either estrus-induced or superovulated animals. The onset of estrus occurred 10-12 hours later (p < 0.01) in G9SYNCH ewes compared with G6SYNCH and G12SYNCH, and the duration of estrus was ∼19 hours greater (p < 0.01) in G9SOV than in G6SOV. The average duration of the NSER procedure was 32.6 ± 1.3 minutes. At least one structure was recovered in 85.7% of synchronized and in 87.5% of superovulated ewes. Viable embryo recovery rates were also similar (p > 0.05) for G6 (1.0 ± 0.3 and 2.5 ± 1.5), G9 (1.3 ± 0.5 and 4.8 ± 2.0), and G12 groups (1.0 ± 0.3 and 4.8 ± 2.3; estrus-synchronized and superovulated ewes, respectively). In conclusion, progestogen pretreatment of different durations and NSER can be employed in Morada Nova ewes, resulting in reasonable viable embryo recovery rates in both estrus-synchronized and superovulated animals. Therefore, both techniques are suitable for use in commercial settings as well as small ruminant conservation programs.

Brucella abortus RB51 ΔleuB expressing Salmonella FliC conjugated gonadotropins reduces mouse fetal numbers: A possible feral swine brucellosis immunocontraceptive vaccine

Population and health management of wildlife is a key to environmental health, domestic herd health, and ultimately public health. Many different methods including: surgical sterilization, poison baits, and sponsored hunting programs have been used in the attempt to control populations of various nuisance animal species. Particular interest has been given to immunocontraception through wildlife vaccination protocols. This study specifically looked at the potential immunocontraceptive and protective properties of a Brucella abortus RB51 ΔleuB vaccine expressing Salmonella typhimurium FliC conjugated to porcine follicle stimulating hormone beta subunit (FSHβ) or gonadotropin releasing hormone (GnRH) DNA sequences. B. abortus RB51 ΔleuB pNS4-TrcD-FliC- FSHβ (RB51LFSHβ) and B. abortus RB51 ΔleuB pNS4-TrcD-FliC-GnRH (RB51LGnRH) were tested in a pilot breeding study with BALB/c mice, and a significant reduction in fertility characteristics was observed in both male and female mice. Ultimately, this study provides support to test these vaccine candidates in feral swine, a destructive invasive species in the United States of America.

125 Influence of length of porcine follicle-stimulating hormone (p-FSH) treatment before ovum pickup on ovarian response and invitro embryo production in Holstein heifers

Ovarian follicle stimulation with exogenous FSH before ovum pickup (OPU) in Bos taurus females is a common practice to increase invitro embryo production (IVP). The optimal stimulatory period length for OPU-IVP, however, has not been definitively ascertained. The objective of the present study, therefore, was to determine the effect of length of the superstimulatory treatment period before OPU on ovarian response and IVP in Holstein heifers. Nonpregnant heifers (n=57) 13.8±0.2 months of age with moderate body condition score (3.0±0.1; scale 1 to 5) were assigned in a completely randomised design to one of the following experimental groups: FSH2d: 200mg of p-FSH (Folltropin-V®, Vetoquinol) distributed in four injections (60, 60, 40, and 40mg) of FSH 12h apart; FSH3d: 200mg of p-FSH distributed in six injections (40, 40, 40, 40, 20, and 20mg) of FSH 12h apart. Timing of follicular wave emergence was synchronized by dominant follicle removal 36h before the first p-FSH injection in all heifers. An intravaginal progesterone (P4) implant (1.38g of P4 CIDR®, Zoetis) was inserted at the time of the first p-FSH injection and removed at the time of OPU, which occurred in all heifers at 44h (Nivet et al. 2012 Reproduction 143, 165-171; https://doi.org/10.1530/REP-11-0391) after the last p-FSH injection. Additionally, follicle number was determined at OPU and classified as small (&amp;lt;6mm), medium (6–10mm) or large (&amp;gt;10mm). Oocytes from follicles of different sizes were pooled by heifer at OPU and then classified and subjected to IVP procedures. Differences between treatment groups were evaluated using generalized linear mixed models (SAS 9.4; SAS Institute Inc.) and data are presented as mean±s.e.m. (Table 1). Lengthening the FSH treatment period resulted in a greater (P=0.01) number of large follicles; however, the number of small, medium-size, and total follicles was not different between groups. Number of total recovered oocytes, viable oocytes, cleaved oocytes, as well as recovery rate, percent viable oocytes, and cleavage rate were not different (P&amp;gt;0.2) between groups. Similarly, the number of blastocysts produced per heifer and blastocyst rate were not different (P&amp;gt;0.9) among groups. In conclusion, lengthening the period of FSH treatment by 1 day increased the number of large follicles at OPU but did not improve overall ovarian response, oocyte recovery, or embryo production. Table 1. Ovarian response and IVP of heifers treated with p-FSH during 2 or 3 days before ovum pickup Response FSH2d (n=28) FSH3d (n=29) P-value Small follicles (n) 5.9±0.6 5.7±0.8 0.83 Medium follicles (n) 17.0±2.4 12.9±1.6 0.18 Large follicles (n) 2.5±0.5 4.5±0.6 0.01 Total follicles (n) 25.4±2.6 23.1±1.8 0.60 Total oocytes (n) 17.0±2.5 13.0±1.2 0.23 Recovery rate (%) 62.6±3.7 56.9±3.1 0.26 Viable oocytes (n) 14.4±2.0 11.3±1.1 0.30 Viable oocytes (%) 85.0±2.4 88.0±3.0 0.31 Cleaved oocytes (n) 8.7±1.6 7.1±1.2 0.62 Cleavage rate (%) 54.7±5.7 54.1±5.7 0.96 Blastocysts (n) 3.2±0.6 3.2±0.7 0.98 Blastocyst rate (%) 20.6±4.0 22.4±3.7 0.97

Consequences of twinning induction to Noemi ewes by a recombinant human follicle-stimulating hormone compared with pituitary-derived porcine follicle-stimulating hormone on follicular dynamics, maternal biochemical attributes, and neonatal traits.

Aim:The aim of this study was to investigate the effectiveness of using recombinant human follicle-stimulating hormone (FSH) compared with pituitary-derived porcine FSH given as one dose or multiple doses on the neonatal traits, follicular dynamics, and maternal blood biochemical constituents in Noemi ewes.Materials and Methods:A 3×2 factorial arrangement was designed utilizing 60 adults Noemi ewes to test the effects of using two sources of FSH (human vs. porcine) in addition to control, either given as a single total dose or six descending doses to provoke twinning. Six treatments (T) were tested (n=10 ewes/T). C1 and C6 served as control ewes given saline as one dose and six doses, respectively; H1 and H6 ewes were given human FSH as one and six doses; and P1 and P6 ewes were given porcine FSH similar to the above treatments. Saline and/or FSH administration were administered at days 8, 9, and 10 of the 10-day controlled internal drug release (CIDR) implant. At CIDR removal, fertile rams were used for natural mating. Blood samples for the assessment of serum metabolites were collected.Results:Twinning increased in FSH-treated ewes than control. However, giving FSH of either source as a single dose resulted in a higher incidence of stillbirths. Pregnancy rates were 30, 40, 50, 60, 70, and 80% in C1, C6, P1, P6, H1, and H6, respectively. Respective percent of ewes delivering twins/multiple birth was 0, 0, 80, 66.7, 71.4, and 87.5%. FSH of human source was more efficient for folliculogenesis than porcine FSH. Administration of FSH increased blood cholesterol, decreased high-density lipoprotein; however, low-density lipoprotein levels were not different than control. Moreover, an interaction (p<0.05) exists between source and type of FSH administration on blood glucose. Six doses of FSH elevated blood protein. Blood albumin decreased by porcine-FSH but not affected by human-FSH. Blood globulins were not different due to source of FSH, whereas giving FSH as six doses increased globulins than in single-dose protocol. Contrariwise, an interaction was found between source and type of FSH administration on elevating the activity of alanine aminotransferase and reducing the activity of aspartate aminotransferase.Conclusion:Administration of human FSH at 180 IU in six descending doses resulted in the best neonatal outcomes and maternal health in Noemi ewes.