FSH Injection Research Articles

146 Single injection of follicle-stimulating hormone before ovum pickup in lactating Holstein donors: Oocyte recovery and embryo production

Lactating donor cows frequently have decreased oocyte quality, lower fertilization rates, and impaired early embryonic development due to their lactational metabolic challenges. Synchronization and stimulation of follicular growth before ovum pickup (OPU) has been used to improve oocyte quality and consequently, embryo production. The objective of this study was to evaluate the effects of a single injection of FSH before OPU on oocyte recovery and embryo production in lactating Holstein donors. During June and July 2020, 22 lactating Holstein donors (open, 40 to 90 DIM, producing >90 lbs of milk) from Ruann Dairy (Riverdale, CA) were randomly assigned to one of two treatments (crossover design). Donors did not receive any injections before OPU when assigned to the No FSH treatment. Treatment 1×FSH consisted of a single intramuscular (IM) injection of 140mg of FSH (Folltropin®, Vetoquinol) 36h after gonadotrophin-releasing hormone (GnRH; Fertagyl, Merck®, 129µg, IM). The FSH consisted of a 3.5-mL IM injection of 400mg of FSH diluted in 10mL of 0.5% hyaluronan (HA). OPU was performed 48 to 52h after FSH. All donors received both treatments on a 14-day interval. The recovered oocytes were fertilized with the same sexed female-sorted semen in both rounds (3 different sires were used). OPU, oocyte classification, IVM, IVF, and culture (IVC) were performed as described by Demetrio et al. (2020 Anim. Reprod. 17, e20200053). All oocytes went into IVM, except for degenerated occytes. The number of 4-cell (or more) embryos on Day 3 of IVC divided by the number of oocytes in IVC after IVF is defined as the cleavage rate. The number of blastocysts (early to hatched) on Day 7 of IVP divided by the number of oocytes in IVC after IVF is defined as the blastocyst rate. Poisson-normal (count data) and Logistic-normal (proportion data) models were used to analyse the data. Treatment, donor (random effect), and sire were included in the models. The results are summarised in Table 1. Oocyte recovery and embryo production were highly donor dependent. There were no differences in the number of recovered oocytes among treatments. Stimulation of the follicular growth before OPU with one single injection of FSH diluted in 0.5% HA 36h after GnRH improved oocyte quality, cleavage rates, blastocyst rates, embryo quality, and the total number of embryos per OPU in lactating Holstein donors. Table 1. Oocyte recovery, cleavage rate, and embryo production results Treatment OPU Oocytes per donor Grade 1 and 2 oocytes Cleavage rate (%) Blastocyst rate (%) Total embryos per OPU Grade 1 embryos per OPU No FSH 22 18.2 30a 69a 22a 3.4a 1.6a 1×FSH 22 17.5 34b 77b 34b 5.3b 3.3b a,bValues with different superscripts in the same column differ (at least P<0.05).

Ovarian responses and FSH profiles at superovulation with a single epidural administration of gonadotropin in the Thai-Holstein crossbreed.

The conventional method of ovarian superstimulation requires multiple injections of gonadotropins which is time-consuming and may be stressful for the cows. This study was designed to determine whether a single epidural injection of FSH (EI group) would induce the superovulatory response in the Thai-Holstein crossbreed and evaluate FSH plasma hormone concentrations. Eight cows (replication = 3; n=24) were assigned to one of 2 treatments in switch back design. Control group (n=12): cows were received 400 mg FSH twice daily by intramuscularly for 4 days (80, 80, 60, 60, 40, 40, 20 and 20 mg), EI group (n=12): cows were received 400 mg FSH by single epidural injection. Data were collected in term of ovarian follicle responses, superovulatory responses, ova/embryo collection. FSH concentrations were examined using ELISA. The total follicular responses during oestrus were not different between treatments; however, the large follicles were less frequent (P < 0.01) while the medium follicle sizes were higher (P < 0.05) in the EI group. The plasma concentration of FSH in EI was dramatically increased within 2 hours before decreasing sharply thereafter (P < 0.01) and did not remain above baseline after 10 hours of administration. The embryo quality was better in the control than the EI groups (P < 0.05). Interestingly, the number of ovulation cysts in the EI group was 50%. The ovarian responses and embryo quality in the cows with cysts were worse compared with the non-cyst groups (P < 0.05). In conclusion, alternative protocols decreased the superovulatory response and increased poor embryo quality in Thai-Holstein crossbred. Also, the incidence of ovarian follicular cysts is higher in the EI group.

71 Effect of follicle-stimulating hormone dose and circulating progesterone before ovum pickup and invitro embryo production in pregnant Holstein heifers

Superstimulation with exogenous FSH before ovum pickup for invitro embryo production (IVP) improves embryo production. However, there is limited information on different FSH dose regimens and the effect of progesterone (P4) concentrations on IVP. Thus, the objective of the present study was to evaluate the effect of FSH dose and circulating P4 on ovarian superstimulation responses, before ovum pickup (OPU) on IVP in pregnant heifers. Pregnant Holstein heifers (n=47) 19.5±0.3 months of age with moderate body condition score (3.3±0.03; scale 1 to 5) were randomly assigned to 2 treatment groups, p-FSH (Folltropin-V, Vetoquinol) 160mg (FSH160) or p-FSH 300mg (FSH300), in a crossover design. Two sessions of OPU were performed on each animal at 48 and 62 days of gestation, with a washout interval between sessions of 14 days. Dominant follicle removal (DFR) was utilised to synchronize follicular wave emergence, and FSH treatments were initiated 36h later. Heifers in both groups received a total of 4 FSH injections 12h apart as follows: FSH160 (48.0, 42.7, 37.3, and 32.0mg) or FSH300 (90.0, 80.0, 70.0, and 60.0mg). All heifers underwent OPU 40h after the last FSH treatment. Blood samples were collected for P4 quantitation at OPU and number of small (&amp;lt;6mm), medium (6–10mm), and large (&amp;gt;10mm) follicles were determined before OPU. Cumulus–oocyte complexes (COCs) recovered were processed and invitro embryo production was performed using standard procedures. Differences between treatment groups were evaluated using generalized linear mixed models, and data are presented as mean±s.e.m. (Table 1). Heifers treated with 300mg of p-FSH had a greater (P&amp;lt;0.05) number of medium-size follicles, total number of follicles, total COCs recovered, number of viable COCs, and number of blastocysts produced. In addition, heifers treated with 300mg of p-FSH had a greater cleavage rate (P=0.03). Circulating P4 was on average 10.1±0.4ng mL−1 and did not differ between gestation days (P=0.12). Furthermore, circulating P4 did not affect (P&amp;gt;0.10) any of the outcomes evaluated, nor was there an FSH dose by circulating P4 interaction (P&amp;gt;0.05). In conclusion, increasing the dose of p-FSH before OPU from 160 to 300mg resulted in an increase in the total number of follicles, number of oocytes, and number of blastocysts produced in pregnant donors without changes in blastocyst rate, regardless of progesterone concentrations at the time of OPU. Table 1. Ovum pickup and invitro embryo production in pregnant heifers treated with different doses of p-FSH Item FSH160 FSH300 P-value Small follicles (n) 5.0±0.6 3.5±0.4 0.02 Medium follicles (n) 14.4±0.8 18.3±0.9 0.0003 Large follicles (n) 2.9±0.3 2.5±0.3 0.28 Total follicles (n) 22.3±0.7 24.3±0.9 0.04 Total oocytes (n) 12.8±0.7 14.7±1.0 0.02 Recovery rate (%) 56.2±2.1 59.7±2.7 0.20 Viable oocytes (n) 11.4±0.7 13.3±1.0 0.02 Viable oocytes (%) 89.9±1.7 88.0±2.4 0.22 Cleavage rate (%) 63.8±4.3 71.5±3.7 0.03 Cleaved oocytes (n) 7.2±0.7 9.3±0.9 0.14 Blastocysts (n) 2.7±0.4 3.6±0.7 0.02 Blastocyst rate (%) 22.0±3.3 22.4±3.6 0.74

12 Artificial insemination and embryo transfer results in ewes during a long daylength period

Sheep are polyestrous, short-day breeders (∼11h of daylength), and exhibit oestrus approximately every 16–17 days during the breeding season, usually in late September to late December in the Northern Hemisphere. Progressive sheep producers often use assisted reproductive techniques such as laparoscopic AI and ovarian hyper-stimulation, embryo collection, and embryo transfer (ET) to increase genetic gain, and strive to have early December lambs to target specific show markets, which dictates AI or ET during the late summer. This field trial compared pregnancy rates following AI or ET in July and August (∼14h of daylength) in southwest Virginia (36–38′12″ N). Ewes (AI, n=83; ET recipients, n=33) were synchronized using a modified Ovsynch protocol involving intravaginal progesterone implants for 14 days, prostaglandin F2α (intramuscular) 48h before expected oestrus, and PG600 (IM) and gonadotrophin-releasing hormone (intramuscular) 52 to 54h and 16h before AI, respectively. Ewes were subjected to AI (frozen/thawed semen) regardless of whether they displayed signs of oestrus, and ewes selected as embryo recipients were subjected to a similar protocol but instead received 1 or 2 embryos (based upon the number of viable embryos produced per embryo donor) 6 days following the AI of the embryo donors. Ovarian hyper-stimulation of the embryo donors (n=13) was enabled by twice-daily FSH injections [totalling 350–455IU of Folltropin V (10–13mL)] for the 4 days before AI. Six days following AI, embryos were recovered surgically from the embryo donors (n=13) and yielded an average (±s.e.m.) of 6.6±1.2 total ova, 4.7±1.1 transferable quality embryos, and 1.9±0.8 unfertilized ova per collection. Pregnancy was detected using transrectal ultrasonography at ∼30 days of gestation and the pregnancy rates were analysed using Chi-squared. There was a tendency (P=0.092) for more pregnancies to be established following ET (22/33; 66%) compared with AI (41/83; 49%). There was no statistical relationship between AI ewes or ET recipient ewes that became pregnant relative to whether they displayed signs of oestrus or not. Embryo transfer was a more successful approach to produce pregnancies in ewes compared with AI during long daylength periods in this field trial. Further, ova from hyper-stimulated embryo donor ewes experienced a very high fertilization rate. Future studies will evaluate the ova capability directly via laparoscopic aspiration of ovarian follicles and IVF and evaluation of hyper- and non-hyper-stimulated follicular waves (using transrectal ultrasonography) and endocrine dynamics during different long and short daylength periods. Extending the opportunity to generate embryos and offspring regardless of daylength will assist aggressive sheep producers in reaching their reproductive, financial, and genetic goals.

Pregnancy rate of intrauterine insemination (IUI) - is it affected by inseminated motile sperm count and stimulation protocol?

Objective To compare IUI pregnancy rate by partner and donor sperm according to inseminated motile sperm count and ovulation induction protocol Design Retrospective study by a private IVF center Materials and methods 3,028 IUI cycles (2,284 cycles with partner sperm and 744 cycles with donor sperm) were analyzed. Ovulation induction was done using 5 mg of letrozole (Let) on cycle days 3 through 7, with 250 mcg recombinant hCG injected as instructed after follicle monitoring is done on day 12. A sequential (Seq) stimulation involves Let protocol, followed by 150 IU FSH daily from cycle day 7. Ultrasound monitoring is done on day 10 to determine further medication dosing, GnRH antagonist initiation, and monitoring. A controlled ovarian hyperstimulation (COH) cycle involves daily FSH injections at doses between 150 and 300 IU FSH, with monitoring beginning on day 7 to determine further medication dosing, GnRH antagonist initiation, and monitoring. All IUI procedures were performed approximately 36 hours after trigger. Pre-washed frozen sperm was thawed by agent's thawing protocol. Total motile sperm for insemination was counted after finishing sperm wash process. The statistical analysis was performed using t-test, chi-square, or z-test. A p value of Results IUI pregnancy rate by using partner sperm was significantly lower (n=106, 3.8%) when inseminated motile sperm is Conclusions IUI pregnancy rate is not affected by sperm count if at least 3 million motile sperm are inseminated. Pregnancy rate from natural cycle is low in both partner sperm and donor sperm cycle. There is no significant difference of pregnancy rate between ovulation induction protocols although the pregnancy rate in Let group is slightly lower than that in COH or Seq group.

Synchronization of germinal vesicle maturity improves efficacy of in vitro embryo production in Holstein cows

Germinal vesicle oocytes obtained by ovum pick-up (OPU) on a random day are heterogeneous in terms of chromatin maturity, and those with an intermediate degree of chromatin compaction present higher developmental competence. We previously developed a synchronization protocol combining follicle aspiration and FSH treatment capable of increasing the percentage of oocytes with intermediate chromatin compaction (classified as GV2 oocytes; within progressive stages of chromatin compaction ranging from GV0 to GV3) at the time of OPU. In this study, we tested the capacity of a similar protocol to synchronize oocyte chromatin maturity before OPU, as well as to improve the efficacy of in vitro embryo production (IVP) in Holstein cows. In the first experiment, eight non-lactating Holstein cows were subjected to the D5/4FSH, during which all follicles larger than 2 mm were aspirated and a progesterone intravaginal device was inserted on a random day (day 0). Subsequently, four IM injections of FSH (Folltropin; 40/40/20/20 mg) were administered 12h apart on days 2 and 3, and removal of the progesterone device and OPU were performed on day 5. Of the oocytes recovered by OPU, 83.2% were at the GV2 stage. In a second experiment, eighteen non-lactating Holstein cows (Synchro group) were subjected to the D5/4FSH protocol followed by IVM/IVF, and embryo production was compared with that of other seventeen cows submitted to OPU on a random day followed by IVM/IVF (Control group). Blastocyst rate in relation to total oocytes recovered was higher in the Synchro group (37.9%) compared to the Control group (21%; P < 0.05). The percentage of good quality blastocysts morphologically selected for freezing and later transfer in relation to the total number of oocytes recovered tended to be higher in the Synchro group (27.68%) compared to the Control group (14.34%; P = 0.1). These data suggest that synchronization protocols increasing the percentage of GV2 oocytes in the population subjected to IVM/IVF can improve the efficacy of embryo in vitro production in cattle.

Follicle priming by FSH and pre-maturation culture to improve oocyte quality in vivo and in vitro

Nowadays there is strong demand to produce embryos from premium quality cattle, and we can produce embryos using oocytes collected from living premium animals by ovum-pick up (OPU) followed by in vitro fertilization (IVF). However, the developmental competence of IVF oocytes to form blastocysts is variable. The developmental competence of oocytes depends on the size and stages of follicles, and follicle-stimulating hormone priming (FSH-priming) prior to OPU can promote follicular growth and improve the developmental competence of oocytes. Furthermore, following the induction of ovulation using an injection of luteinizing hormone or gonadotropin-releasing hormone after FSH-priming, we can collect in vivo matured oocytes from ovulatory follicles, which show higher developmental competence than oocytes matured in vitro. However, the conventional protocols for FSH-priming consist of multiple FSH injection for 3–4 days, which is stressful for the animal and labor-intensive for the veterinarian. In addition, these techniques cannot be applied to IVF of oocytes collected from bovine ovaries derived from slaughterhouses, which are important sources of oocytes. Here, we review previous research focused on FSH-priming, especially for collecting in vivo matured oocytes and a simplified method for superstimulation using a single injection of FSH. We also introduce the previous achievements using in vitro pre-maturation culture, which can improve the developmental competence of oocytes derived from non-stimulated animals.

Using Anti-M&amp;#252;llerian Hormone (AMH) as a Predictor of Ova Production for Bovine Embryo Transfer

The objective of this study was to determine the relationship of anti-Müllerian hormone (AMH) to ova production in various breeds of cattle in an embryo transfer program. Various factors that were evaluated included breed type, age, weight, body condition and flush history as to their influence on total ova recovery. The superovulation regimen called for CIDR (Controlled Internal Drug Release) insertion and a 2 cc injection of Combo (25 mg progesterone and 1.25 mg of estradiol per mL (2 cc IM injection)) on day 0. Day 4, FSH treatments were initiated in both AM and PM with decreasing amounts over the next four days. Day 6, along with FSH, prostaglandin was given in the AM and PM. On Day 7, a final FSH injection and CIDR removal were done in the AM. This resulted in estrus and AI on day 8 and collection of ova on day 15. Results were drawn from analyzing 369 animals; Angus n = 25, Black Brangus n = 43, Red Brangus n = 53, Brahman n = 103, Beefmaster n = 112, and Wagyu n = 33. Age of donors ranged from 1.6 years to 15.4 years at collection with an average age of 7.16 years of age. Analysis of results demonstrated that age clearly had a significant (P 0.05) on ova production. More importantly, we observed the total ova production was significantly (P < 0.05) associated with the AMH concentration (the greater the AMH concentration, the greater number of ova per flush).

107 Follicular wave synchronization and FSH stimulation prior to ovum pickup for invitro embryo production

Invitro embryo production (IVP) has become a reliable alternative for genetic improvement in beef and dairy herds. Maximizing embryo yield and pregnancy per donor are key factors. The aim of this study was to compare ovum pickup (OPU) yields, developmental competence of cumulus-oocyte complexes (COCs), and pregnancy rates from Angus donors in a commercial IVP setting. Donors (&amp;gt;4-year-old pluriparous open dry cows) were handled under the same feeding and environmental conditions. Treatment groups were organised as follows: Group 1: no synchronization (SYNCH; n=5); Group 2: SYNCH with no superstimulation (SOV; n=5); Group 3: SYNCH + SOV (n=5) and OPU 36h after last FSH injection; Group 4: SYNCH + SOV (n=5) and OPU 48h after last FSH injection; and Group 5: SYNCH + SOV (n=5) and OPU 72h after last FSH injection. Follicular waves in groups 2, 3, 4, and 5 were synched by gonadotrophin-releasing hormone (GnRH), prostaglandin F2α (PGF), and controlled internal drug release (CIDR). No pre-synch was used. Injections of FSH (pFSH=180mg, Folltropin) were performed IM twice a day, for three days. A minimum of three replicates were performed for each donor. A Mindray DP30V equipped with a micro-convex transducer 5.0-8.5MHz probe, disposable 20-gauge needle, and a flow rate of 15mL min−1 were used for OPU. All visible follicles (Foll) were punctured and retrieved into a 50-mL 36°C warmed tube with media (phosphate-buffered saline, bovine serum albumin (BSA), and heparin). Viable oocytes were classified according to IETS guidelines. The COCs were matured in 100µL of M199 medium supplemented with ALA-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µgmL−1), epidermal growth factor (50ngmL−1), oFSH (50ngmL−1), bLH (3μgmL−1), cysteamine (0.1mM), and 10% fetal bovine serum (FBS) for 22 to 24h. Fertilization (Day 0) was carried out using highly fertile sires selected by discontinuous 40%/80% layers (PureSperm) and diluted to a final concentration of 1×106 spermmL−1. Matured oocytes were fertilized in 50µL of modified synthetic oviductal fluid (SOF) media supplemented with fructose (90µgmL−1), penicillamine (3µgmL−1), hypotaurine (11µgmL−1), and heparin (10µgmL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in 50-µL drops of SOF-BSA for 7 days. On Day 3.5, 2% of FBS was added. On Day 7, fresh transferable (grade 1 and 2, IETS standards) blastocysts were implanted into synchronized recipient cows. Around Day 30, ultrasound diagnosis was performed to determined pregnancy rate (PR). We used ANOVA for comparisons of mean values and X2 test for proportions, α=0.05 (Table 1). In conclusion, synchronization, FSH stimulation, and 48-h coasting before OPU in Angus cows increased the number of collected viable oocytes and embryo development rates. More transferrable embryos and higher rates of PR per OPU were obtained using 36- and 48-h coasting, respectively. Table 1.Follicles (Foll), viable oocytes, cleavage, blastocysts, and pregnancy rates (PR) in Angus cows after ovum pickup (OPU) or invitro embryo production (IVP) SYNCH1 FSH Coasting Foll/OPU Oocytes/OPU Viable oocytes Cleavage,% Embryos,% Embryos/OPU PR,% PR/OPU NO NO NO 10.8a 9.0a 8.9a 63a 25a 2.3a 45a 1.0a YES NO NO 10.1a 8.4a 8.1a 73b 27b 2.2a 51b 1.1a YES YES 36H 16.5b 13.8b 13.6b 70c 29b 4.0b 46a 1.8b YES YES 48H 16.1b 13.4b 13.3b 69c 28b 3.8c 52b 2.0b YES YES 72H 15.6b 13.0b 12.6b 64a 22c 2.8d 55b 1.6c a-dValues with different superscripts in the same column differ (P&amp;lt;0.05). 1SYNCH=synchronized.

176 Synchronisation of follicle wave emergence prior to superstimulation with purified FSH for ovum pickup affects blastocyst rate in pregnant Holstein heifers

The timing of initiation of superstimulatory treatments relative to follicle wave emergence has been shown to affect ovulatory response and invivo embryo production. The significant increase of invitro embryo production (IVP) and the possibility of using pregnant animals as oocyte donors has created the need to optimise superstimulatory treatments for IVP in pregnant cattle. Thus, the objective of the present study was to evaluate the effect of synchronisation of follicle wave emergence before superstimulation for ovum pickup (OPU) and IVP in pregnant heifers. Pregnant (47-62 days of gestation) Holstein heifers (n=28) 19.5±0.3 months of age were assigned in a completely randomised design to one of two groups: synchronisation of follicular wave emergence by dominant follicle removal (DFR; all follicles &amp;gt;6mm) or untreated control (no DFR). Superstimulatory treatments were initiated 36h after DFR or at random stages of the follicular wave in the no-DFR group and consisted of the administration of 160mg of purified FSH (Folltropin-V, Vetoquinol) over four injections 12h apart as follows: 48.0, 42.7, 37.3, and 32.0mg. Ovum pickup was performed in all heifers 40h after the last purified FSH injection. Heifers were subjected to OPU for oocyte recovery, and the number of follicles was determined. Recovered oocytes were processed in groups by treatment, and IVP was performed. Differences between treatment groups were evaluated using generalised linear mixed models. Results are presented in Table 1 and are expressed as means±s.e.m. for data collected at the time of OPU or as proportions for embryo production results. The number of small follicles (&amp;lt;6mm) at the time of OPU was greater in the no-DFR group than in the DFR group (P=0.04). Conversely, there were no differences between treatments in the number of medium follicles (6-10 mm; P=0.17), large follicles (&amp;gt;10 mm; P=0.11), total follicles (P=0.93), total number of recovered oocytes (P=0.4), or number of viable oocytes (P=0.53). The mean oocyte percentage recovery rate was not different between heifers in the DFR (53.6±4.7%) and no-DFR (56.5±4.7%) groups (P=0.52). Both cleavage and blastocyst rate were greater (P&amp;lt;0.008) in the DFR group than in the no-DFR group; as a result, the number of transferable embryos per animal was 5.6 in the DFR group and 2.8 in the no-DFR group. In summary, initiation of superstimulatory treatments at the time of follicle wave emergence improves cleavage and blastocyst rates, thus leading to greater embryo production. Table 1.Ovarian response and embryo production in pregnant heifers superstimulated with or without synchronisation of follicle wave emergence Variable DFR No DFR Small follicles, n 8.1±1.2A 12.1±1.8B Medium follicles, n 18.3±1.3 13.7±2.0 Large follicles, n 2.4±0.6 1.4±0.4 Total follicles, n 28.8±1.4 27.2±2.2 Total oocytes, n 15.4±1.5 16.0±1.9 Viable oocytes, n 13.7±1.5 13.4±1.8 Cleavage rate,% (n) 77.1 (192)A 64.4 (188)B Blastocyst rate,% (n) 40.6 (192)A 20.7 (188)B A,BMeans within a row with different superscripts differ (P&amp;lt;0.05).

Factors affecting embryo production in superovulated Bos taurus cattle.

Despite a long history of bovine superovulation research, significant commercial applications did not start until the early 1970s. For some 20 years thereafter, superovulation represented the primary tool for the production of cattle embryos. In the early 1990s, commercial invitro production (IVP) was initiated in cattle. Although ovum pick-up and IVP are now commercially practiced on a wide scale, superovulation and embryo recovery by flushing remain a widespread and very effective approach to the production of cattle embryos. This review covers both the history and the effects of multiple factors on superovulation in Bos taurus cattle. There are three general protocols for suitable pre-FSH programming of donors so that gonadotrophin-responsive follicles are available. Superovulation protocols vary widely based on the FSH source, the diluent used, the number and timing of FSH injections and the timing and utilisation of various prostaglandins, controlled internal progesterone releasing devices, gonadotrophin-releasing hormone, and other means of controlling follicular development and ovulation. The number of oocytes that can be stimulated to grow and ovulate within any given donor can be estimated by either ultrasound-guided sonography or by measuring concentrations of anti-Müllerian hormone in the blood. Animal-related factors that can influence the efficacy of superovulation include cattle breed, age, parity, genetics, lactational status and reproductive history. In addition, nutrition, stress, season, climate, weather and several semen factors are discussed.

Characterization and control of oocyte large-scale chromatin configuration in different cattle breeds

Differences in reproductive physiology between cattle breeds may help to explain distinct responses to assisted reproductive techniques and to define breed-specific protocols with improved efficiency. Germinal vesicle (GV) stage oocytes are characterized by increasing levels of chromatin compaction enclosed within the nucleus (graded from GV0 to GV3), associated with different developmental competence. The first objective of this study was to characterize chromatin configuration of GV stage oocytes recovered by OPU at random days of the estrous cycle from Nelore (Bos indicus) and Holstein (Bos taurus) cows. In Nelore 90% of the oocytes presented advanced stages of chromatin compaction associated with higher developmental competence (GV2 and GV3), while in Holstein, only 65% of the oocytes were at these stages. Then, aiming to obtain a more homogeneous population of oocytes in Holstein, we tested two synchronization protocols combining aspiration of all visible follicles at a random day (day 0), two IM injections of FSH 12 h apart on day 2, and OPU on day 4 (OPU/D4) or 5 (OPU/D5). The protocol OPU/D4 provided around 45% of the oocytes with low chromatin compaction (GV1), while the protocol OPU/D5 provided 70% of the oocytes at GV2 and 20% at GV3. Finally, we assessed the effects of a culture system known to prevent meiotic resumption on chromatin configuration of the GV2 enriched oocyte population obtained with the protocol OPU/D5. After 9 h of culture most oocytes transited from GV2 to GV3, with 90% of the oocytes at GV3 stage. This study demonstrates differences between Nelore and Holstein cows regarding patterns of chromatin configuration that may account for their different performance in IVM/IVF. In addition, it provides novel references for the design of protocols aiming to regulate oocyte quality before IVM for the optimization of IVF outcomes.

Effect of FSH dosage on the number and quality of Pesisir cattle embryos

This study investigates the effect of FSH supplementation on the resulting number and quality of super-ovulated Pesisir cattle embryos. This study aims investigates the effect of FSH supplementation on the resulting number and quality of super-ovulated Pesisir cattle embryos. After progesterone therapy fifteen Pesisir cattle were each given five injections of FSH followed by AI with Simmental semen. Embryos were flushed out on the sixth to eighth day after AI and counted and graded under a microscope. Cows given 16 ml and 12 ml of FSH produced the highest percentage of A quality embryos (22% and 20% respectively) while no high-quality embryos were collected from those given 14 ml FSH. 12 ml FSH treatment resulted in the highest number of B quality embryos (20%) while a14 ml dose produced less than 7%. One-third of the embryos resulting from 14 ml FSH were C grade but 12 and 16 ml dosages resulted in no embryos of this grade. The highest numbers of degraded embryos were found from cows given 12 ml (60%), while the lowest from cows given 14 ml (33.33%). The percentage of the recovered ovum that remained unfertilized from cows treated with 16 ml, 14 ml, and 12 ml were 33.3%, 66.7%, and 0% respectively. While 14ml dosages of FSH resulted in the highest number of embryos, the higher embryo from a 16 ml FSH suggests that this dosage will produce superior results.

Corifollitropin alfa for ovarian stimulation in in vitro fertilization: a systematic review and meta-analysis of randomized controlled trials

To evaluate the effectiveness of corifollitropin alfa in improving the success of IVF. Systematic review and meta-analysis. Not applicable. Infertile women undergoing conventional IVF or intracytoplasmic sperm injection (ICSI). Randomized controlled trials (RCTs) of infertile women undergoing a single IVF/ICSI cycle with either corifollitropin alfa or a conventional ovarian stimulation protocol based on daily injections. The review protocol was registered in PROSPERO before starting the data extraction (CRD42018088605). Primary outcomes were live birth rate and/or ongoing pregnancy rate. Clinical pregnancy rate, miscarriage rate, multiple pregnancies, number of oocytes and embryos obtained, cancellation rate, and rate of ovarian hyperstimulation syndrome and ectopic pregnancy were considered as secondary outcomes. Eight randomized controlled trials were included; 2,345 women were assigned to the intervention group and 1,995 to the control group. The analysis of 4,340 IVF cycles did not reveal any difference in live birth rate and/or ongoing pregnancy rate between groups (risk ratio [RR], 0.92; 95% confidence interval [CI], 0.80-1.05). Similarly, no difference was found in clinical pregnancy rate (RR, 0.96; 95% CI, 0.88-1.05; I2 = 0%), miscarriage rate (RR, 0.94; 95% CI, 0.71-1.25; I2 = 0%), or multiple pregnancy rate (RR, 1.22; 95% CI, 0.99-1.50; I2 = 0%). Also, the rates of cycle cancellation, ovarian hyperstimulation syndrome, and ectopic pregnancy were similar in both groups. Sensitivity and subgroup analyses did not provide statistical changes to pooled results. Corifollitropin alfa seems to be an alternative for daily recombinant FSH injections in normal and poor responder patients undergoing ovarian stimulation in IVF/ICSI treatment cycles.

Combined effect of heat and nutritional stress on superovulation of Malpura ewes in a semi-arid region

Sheep reared in hot semi-arid environments are generally exposed to heat and nutritional stress in some seasons of the year, which affects both production and reproduction. To assess the effect of high ambient temperature and feed scarcity on superovulation, 16 adult Malpura ewes were randomly divided into two groups of 8 animals each. G1 (control) was kept under a shed and offered a maintenance diet, and G2 (combined stress) was subjected to both nutritional (30% less of maintenance diet) and heat (38–44 °C for 6 h/day) stress. Ewes were superovulated without estrus synchronization by a combination of single injection of 200 IU eCG and 8 injections of FSH (Folltropin-V) at 12-h intervals in tapering doses of 5 mg/kg body weight, starting from the day 7 of natural estrus. eCG was given with the first injection and PGF2α (10 mg) was given with the second last FSH injection. G2 increased respiration rate and rectal temperature (P < 0.01), and blood urea level (P < 0.05), whereas it decreased average daily gain, plasma T4 concentration (P < 0.01) and body weight (P < 0.05). Plasma estradiol level was lower (P < 0.05) in G2 ewes as compared to control (G1) ewes. However, the number of ewes showed a superovulatory response (88 vs 66% ewes ≥ 3 corpus luteum), ovulation rate (8.75 vs 5.88) and embryo production (5.5 vs 3.9) decreased, and the number of large follicles (anovulation) increased (1.0 vs 2.14) in G2 ewes. G2 had a comparable effect on the superovulatory response compared to control ewes although physiological changes occurred as an adaptive mechanism to stress. Therefore, the well-adapted cyclic sheep of the semi-arid region may be used for superovulation despite the stressful condition of heat exposure and nutritional insufficiency.

Small Molecule Follicle-Stimulating Hormone Receptor Agonists and Antagonists.

The follicle-stimulating hormone receptor (FSHR) has been targeted therapeutically for decades, due to its pivotal role in reproduction. To date, only purified and recombinant/biosimilar FSH have been used to target FSHR in assisted reproduction, with the exception of corifollitropin alfa; a modified gonadotropin in which the FSH beta subunit is joined to the C-terminal peptide of the human choriogonadotropin beta subunit, to extend serum half-life. Assisted reproduction protocols usually entail the trauma of multiple injections of FSH to initiate and promote folliculogenesis, which has prompted the development of a number of orally-available low molecular weight (LMW) chemical scaffolds targeting the FSHR. Furthermore, the recently documented roles of the FSHR in diverse extragonadal tissues, including cancer, fat metabolism, and bone density regulation, has highlighted the potential utility of LMW modulators of FSHR activity. Despite these chemical scaffolds encompassing a spectrum of in vitro and in vivo activities and pharmacological profiles, none have yet reached the clinic. In this review we discuss the major chemical classes of LMW molecules targeting the FSHR, and document their activity profiles and current status of development, in addition to discussing potential clinical applications.

Superovulation in cattle – searching for the optimal dose, alternative routes of administration and a simplified FSH application program

The aim of this article is to set out certain possibilities related to the simplification of the superovulation protocols (SOV) in cattle. The traditional protocols, which have been in use for decades, include as standard several injections of FSH preparations. Due to the widespread increase in the importance of the animals’ welfare, alternative options for its administration are now being sought. Appropriate selection of gonadotropin as well as the dose and ratio of hormones makes it possible to limit undesirable effects such as prolonged ovarian stimulation. Adjuvants such as polyvinylpyrrolidone, polyethylene glycol, hyaluronic acid or aluminium hydroxide enable the extension of the absorption time of FSH preparations, thus reducing the amount of FSH injections. A single deposition of hormones dissolved in aqueous solutions is possible when it is administered via alternative routes, such as subcutaneous or epidural. Reduction of the amount of FSH injections may directly translate into a decrease of the stress level in animals treated by the SOV protocols and also to the improvement of the efficiency of embryo production. A significant risk factor remains the high variability of superovulation. Its effectiveness depends on a number of generally well known individual and environmental factors that should also be considered when joining SOV.

138 Effects of different treatments of donors on the efficiency of embryo production and conception in bovine ovum pickup-in vitro production

An in vitro-produced bovine embryo has a low conception rate compared with that of an in vivo embryo. The present study was conducted to examine the effects of different treatments delivered to donors before ovum pickup (OPU) sessions to improve the conception rate of in vitro-produced bovine embryos. In total, 351 OPU sessions were performed on 138 Holstein and 213 Japanese Black cows from January to December 2017. Donors were divided into 4 groups based on their pretreatment before OPU: (1) single injection of 2.5 AU of FSH 40h before OPU; (2) CIDR insertion on Day 0, injection of 2mg of oestradiol benzoate on Day 1, 4 injections of FSH (each 2.5 AU) every 12h beginning from Day 5 to 7, followed by removal of CIDR and OPU on Day 9; (3) injection of 50μg of gonadotropin-releasing hormone 72h before OPU; or (4) no pretreatment. The collected cumulus-oocyte complexes were matured for 22h in 25mM of HEPES buffered TCM-199 supplemented with 5% newborn calf serum and 0.02 AU mL−1 FSH. After 6h of gamete co-culture (5.0×106 sperm mL−1), the presumptive zygotes were washed and the remaining cumulus cells were denuded by pipetting. The presumptive zygotes were then cultured in KSOMaa supplemented with 5% newborn calf serum for 9 days in a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). Blastocyst formation rates were analysed 9 days after insemination, and the formed blastocysts were transferred to oestrous synchronized recipients on the seventh or eighth day after oestrus. The data were analysed by Chi-squared test with Yates correction. The average numbers of collected oocytes were 57.7±17.4 (n=136), 25.3±12.8 (n=20), 28.8±12.5 (n=18) and 24.3±12.9 (n=177) in groups 1 to 4, respectively. Groups 1 and 2 showed significantly (P&amp;lt;0.01) high percentages of Grade-1 oocytes (52.1 and 49.6%, respectively) compared with groups 3 and 4 (37.3 and 39.9%, respectively). The proportion of blastocysts in groups 1 (38.6%) was significantly different compared with that in groups 2 (32.1%) and 4 (35.3%), but the difference was insignificant in the case of group 3 (36.4%). The conception rates in groups 1 (43.5%, n=868) and 2 (59.1%, n=44) were significantly (P&amp;lt;0.05) higher than those in groups 3 (35.1%, n=57) and 4 (34.9%, n=768). These results suggest that although the efficiency of embryo production did not differ largely between donors pretreated with 4 FSH injections and those without any pretreatment, the conception rate in donors pretreated with 4 FSH injections was significantly higher than that in donors without pretreatment. Moreover, donors pretreated with a single injection of FSH showed significantly high efficiency of embryo production and conception rate than donors without pretreatment.

199 The effect of different doses of follicle-stimulating hormone during superovulation on ovarian function in dairy cattle

Cattle with an unknown antral follicle count (AFC) during follicular waves respond to different FSH doses during superovulation in a curvilinear fashion with the highest doses decreasing or not increasing ovulatory follicle number, number of transferable embryos, and number of corpora lutea (CL). These observations support the hypothesis that heifers treated with different FSH doses during superovulation will reach a superovulation maximum (SOVmax) and that doses exceeding SOVmax are excessive, resulting in a decrease in ovarian function. To test this hypothesis, we used eight 12-month-old Holstein heifers with a low AFC (&amp;lt;10 follicles ≥3mm in diameter, index for small ovarian reserve). These 8 heifers were subjected to 4 different superovulations with a 21-day intervals between each superovulation. During each superovulation, which began on Day 1 of a synchronized oestrous cycle before selection of a dominant follicle, 2 of the 8 heifers were treated (twice-daily FSH injections×4 days) with 20, 40, 80, or 120mg of Folltropin-V (Vetoquinol, Lure, France). At the end of the study, the same 8 heifers had been superovulated 4 times with each of the 4 FSH doses. To enhance ovulation rate in response to hCG, each superovulated heifer was injected with 3 different injections of prostaglandin F2α (PGF2α) spaced 12h apart to induce luteolysis of the original CL. Two PGF2α injections were given after each FSH injection on the last day of FSH treatment and the last PGF2α injection was given after the hCG (2500IU) injection, which was 12h after the last FSH injection. Daily ultrasonography was used to measure AFC and number of ovulatory follicles and CL during the study. Results of t-test analysis showed that at time of hCG, AFC was greater (P&amp;gt;0.05) for heifers treated with the 40-mg (mean±s.e.m., 27±3.7) v. 20-mg (17.9±2.5) FSH dose, but the response to higher FSH doses (27.9±4.4; 27±3.3) did not differ from the response to the 40-mg dose. Number of ovulatory follicles was greater (P&amp;lt;0.05) for heifers treated with the 40-mg (17.5±2.3) v. 20-mg (10.1±1.4) FSH dose, but the response to higher doses (18.9±3.3; 17.3±2.4) did not differ from the response to the 40-mg dose. At Day 7 post-hCG, CL number was greater (P&amp;lt;0.05) for heifers treated with the 40-mg (13.8±2.9) v. 20-mg (7.4±1.4) dose, but response to higher doses (9.9±3.2; 9.1±2.3) did not differ from the response to the 40-mg dose. Ovulation rate (ovulatory follicle number divided by CL number) was greater (P&amp;lt;0.05) for heifers treated with the 40-mg (79±0.08%) v. 80-mg (52±0.09%) or 120-mg (53±0.09%) dose, but similar to that of heifers treated with 20-mg (73±0.09%) FSH dose. These results support the conclusion that the 40-mg FSH dose achieves SOVmax and FSH doses greater than SOVmax in cattle with a low AFC and small ovarian reserve are excessive and detrimental to ovarian function. This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2017-67015-26084 from the USDA National Institute of Food and Agriculture (Washington, DC).

196 Embryo production using follicle-stimulating hormone (FSH) or FSH+equine chorionic gonadotropin in beef donors

The objective of the present study was to evaluate the superovulatory response and embryo production in beef donors using 8 twice-daily injections of FSH or an alternative protocol in which the last 4 FSH injections were replaced with a single injection of eCG. In Exp. 1, 12 mature Bonsmara donor cows, with a body condition score between 3 and 4 (1 to 5 scale) were superstimulated twice every 46 days in a crossover design (i.e. in each experiment all cows received 2 treatments and the 2 treatments were equally represented in each replicate). On Day 0 a.m., all donors received an intravaginal device with 1.2g of progesterone (Diprogest 1200®, Zoovet, Santa Fe, Argentina), along with 50mg of progesterone i.m (Progestar®, Zoetis, Buenos Aires, Argentina) and 5mg of oestradiol-17β (17βOestradiol®, Rio de Janeiro, Argentina) IM. On Day 4 a.m., the superstimulatory treatments were initiated and donors in the control group received 8 applications of FSH (Folltropin-V, Vetoquinol, Lure, France), IM (total dose: 300mg NIH-FSH-P1) in a twice-daily decreasing dosage schedule over 4 days (i.e. 60, 60, 50, 50, 30, 30, 10, and 10mg, respectively). Donors in the FSH+800 eCG group received only the first 4 applications of FSH (i.e. 60, 60, 50 and 50mg, respectively) and on Day 6 a.m. they received 800IU of eCG (Novormón®, Zoetis) IM in a single dose. All donors received 500μg of cloprostenol (Ciclase DL®, Zoetis) IM on Day 6 a.m. and p.m., and the intravaginal devices were removed on Day 7 a.m. All cows also received 100μg of gonadorelin acetate (GnRH, Gonasyn gdr, Zoetis) on Day 8 a.m. and were inseminated with frozen-thawed semen from 2 bulls 12 and 24h later. On Day 15, ova/embryos were collected and evaluated according to the IETS standards. The data were analysed by GLMM (Infostat, 2018). In Exp 2., 18 Bonsmara donors with similar conditions as those in Exp. 1 were superovulated twice in a crossover design. Cows in both groups received similar treatments to those in the FSH+eCG treatment of Exp. 1, except that the total dosage of FSH was 200mg (i.e. 60, 60, 40, and 40mg, respectively) and the eCG given on Day 6 a.m. was either 600IU (group 1) or 800IU (group 2). In Exp 1., the FSH (control) group had a higher (P&amp;lt;0.01) number of fertilized oocytes, but there were no differences in the other end points evaluated (Table 1). In Exp. 2, no differences were found between FSH+800 eCG and FSH+600 eCG groups in any of the parameters evaluated. In conclusion, the replacement of the last 4 injections of FSH by a single dose of eCG decreases the number of treatments required in a superovulation program without negatively affecting the production of transferable embryos. Table 1.Embryo production (means±s.e.m.) in Bonsmara donors treated with FSH or FSH+eCG