Single Injection Of FSH 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).

145 Comparison of single to multiple injections of follicle-stimulating hormone before ovum pickup in Holstein heifers: Oocyte recovery and embryo production

The demand for invitro-produced embryos from heifers with high genetic merit has increased over time. Synchronization and stimulation of follicular growth before ovum pickup (OPU) has been used to improve oocyte quality and, consequently, embryo production. Multiple injections involve extra labour and stress for both personnel and cattle. The release of FSH can be prolonged by using 0.5% hyaluronan (HA) as a diluent, allowing a decrease in the number of injections. The objective of this study was to compare oocyte recovery and embryo production between single or multiple injections of FSH before OPU of Holstein heifers. During April and May 2020, 20 Holstein heifers (8 to 15 mo old) from Ruann Dairy (Riverdale, CA) were randomly divided and submitted to two different treatments (crossover design). Gonadotrophin-releasing hormone (GnRH; Fertagyl®, Merck, 129µg, IM) was given to synchronize the follicular wave emergence. Treatment 1×FSH consisted of a single intramuscular (IM) injection of 100mg of FSH (Folltropin®, Vetoquinol) 36h after GnRH. The FSH consisted of a 2.5-mL injection of 400mg of FSH diluted in 10mL of 0.5% HA. OPU was performed 48 to 50h after FSH. Treatment 5×FSH consisted of 100mg of FSH divided into 5 equal IM injections (10-14h intervals) 36h after GnRH. The FSH consisted of 5×1-mL injections of 400mg of FSH in 20mL of saline. OPU was performed 18 to 20h after the last FSH injection. All donors received both treatments at a 14-day interval and the recovered oocytes were fertilized with the same sexed female-sorted semen in both rounds. 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 oocytes. 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 summarized in Table 1. There were no differences between the two treatments on the number of oocytes recovered per OPU (total and grade 1 and 2), percentage of grade 1 and 2 oocytes, cleavage rate, blastocyst rate and number of embryos (total and grade 1). Oocyte recovery and embryo production are highly donor dependent. Stimulation of the follicular growth before OPU with one single injection of FSH diluted in 0.5% HA 36h after GnRH can be efficiently used for IVP in Holstein heifers, without decreasing the number of oocytes recovered and/or embryos produced with the advantage of reducing labour and stress of handling cattle. Table 1. Number and quality of oocytes and cleavage and blastocyst rates Treatment OPU Oocytes per donor Grade 1 and 2 oocytes (%) Cleavage rate (%) Blastocyst rate (%) Total embryos per OPU Grade 1 embryos per OPU 1×FSH 20 17.0 45.7 84 39.8 6.2 3.8 5×FSH 20 19.9 46.5 82 35.6 6.3 4.0

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.

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<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<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.

Respons Superovulasi Sapi Peranakan Ongole terhadap Penyuntikan Tunggal Follicle Stimulating Hormone ke dalam Ruang Epidural (SUPEROVULATION RESPONSES IN ONGOLE CATTLE CROSSBREED TREATED WITH A SINGLE EPIDURAL INJECTION OF FOLLICLE STIMULATING HORMONE)

Super-ovulation is conventionally performed by injection of FSH twice daily for four days. Thistreatment needs frequent attention by farm-personnel and relatively increases the possibility of failuresdue to mishandling and errors in administration of the treatment. This study was conducted to evaluatethe responses of superovulation trough a single injection of FSH into epidural space in ongole cattlecrossbreed. In Experiment 1, a combination of single dose injection of FSH was applied into epidural spaceplus intramuscular (epi+i.m group) compared to the group of intramuscular injection of FSH, which wastreated twice daily for four days (intramuscular group), using equal total dose of FSH 400 mg. Superovulationresponse of epi+im group (n=4) was not significantly different compared with intramusculargroup (n=4). In experiment 2, it compared two treatmentof FSH in different concentration(280 mg versus160 mg) in a single dose applied into epidural space. Data of Epi+im group from experiment 1 was used ascontrol. Group of 280 mg FSH (n=4) resulted total collection of ova/embryo and transferable embryos(9,00+2,65 and 3,33+2,52 respectively) was significantly different compared to the 160 mg group (n=4)(which were 2,00+1,26 and 0,00) P <0,05, although they werenot significantly different compared to thecontrol (9,33+5,68 and 3,67+3,21). In conclusion, injection of a single dose of FSH at 280 mg into epiduralspace result in a comparable transferable embryo which similar to the conventional method that appliedintramuscular injection of FSH twice daily for four days.

Acute follicular response to FSH in heifers downregulated long term with a GnRH agonist and with suppressed ovarian follicular growth

The objectives of this study were to (1) ascertain ovarian follicular status in heifers where the pituitary gonadotrope cells producing LH and FSH were downregulated by long-term treatment with a GnRH agonist and (2) characterize the acute superstimulation of follicular growth in response to FSH in heifers where the pituitary was downregulated with a GnRH agonist. At the start of the study, heifers (3 year old) were implanted with GnRH agonist (n = 20) or received no treatment (control, n = 5). After 6 months, follicular growth in heifers implanted with GnRH agonist was restricted to early antral follicles (2–4 mm). At this time, these heifers were allocated to four groups and follicular development was superstimulated with FSH as follows: group 1 (n = 5) and group 2 (n = 5), a conventional FSH protocol with injections over 4 days (Days 0–3) with Group 2 receiving two times the normal dose of FSH; group 3 (n = 5), a single injection of FSH in saline on Day 0; group 4 (n = 5), a single injection of FSH in slow-release polyvinylpyrrolidone on Day 0. Follicular growth in the control heifers (n = 5) was superstimulated using the conventional 4-day FSH protocol. On Day 5, heifers in groups 1 and 2 had similar (P > 0.05) numbers of follicles in the size categories 6 to 7 mm (12.8 ± 3.0 follicles) and 8 to 9 mm (6.5 ± 1.0) as control heifers (6–7 mm, 14.6 ± 2.2; 8–9 mm, 6.2 ± 2.2) and six of the former heifers had follicles 10 mm (3.2 ± 1.4). Also on Day 5, follicular growth for heifers in group 3 was restricted to 4 to 5 mm (11.6 ± 3.0) and 6 to 7 mm (7.6 ± 3.7), whereas four out of five heifers in group 4 had follicles of 6 to 7 mm (8.2 ± 2.3) and three heifers had follicles of 8 to 9 mm (4.6 ± 2.2) and 10 mm (2.2 ± 0.9). Injection of exogenous LH on Day 5 induced ovulation in 9 out of 10 heifers in groups 1 and 2, no heifers in group 3, and 2 of 5 heifers in group 4. Plasma concentrations of progesterone 6 days after ovulation were the same (P > 0.05) for heifers in groups 1 and 2 (8.9 ± 0.9 ng/mL) and control heifers (10.0 ± 0.9 ng/mL). This study has shown that heifers treated chronically with a GnRH agonist have suppressed ovarian follicular growth but are able to respond to acute superstimulation with FSH. Furthermore, follicles in heifers treated with a GnRH agonist undergo morphologically normal growth in response to FSH and can ovulate and develop into a CL. The long-term GnRH agonist–treated heifer provides a practical model for repeated ovarian follicular superstimulation, multiple ovulation, and embryo transfer, without the need to control stage of the estrous cycle.

Interspecies embryo transfer in camelids: the birth of the first Bactrian camel calves (Camelus bactrianus) from dromedary camels (Camelus dromedarius)

Interspecies embryo transfer is a possible approach that can be used to conserve endangered species. It could provide a useful technique to preserve the Iranian and wild Bactrian camels, both of which are threatened with extinction. In the present study, one Bactrian camel was superovulated using decreasing doses of FSH (60, 40, 30, 30, 20, 20 mg, b.i.d.; Folltropin-V; Bioniche, London, ON, Canada) for 6 days, followed by a single injection of FSH (20 mg, i.m.) on Day 7. Daily ovarian ultrasonography was performed until most of the growing follicles had reached a mature size of 13-17 mm, at which time the camel was mated twice, 24 h apart, with a fertile male Bactrian camel. At the time of first mating, female camels were given 20 microg, i.v., buserelin (Receptal; Intervet, Boxmeer, The Netherlands). One day after the donor camel had been mated, the dromedary recipients (n = 8) were injected with 25 mg, i.v., porcine LH (Lutropin-V; Bioniche) to induce ovulation. Embryos were recovered on Day 8.5 after the first mating and transferred non-surgically into recipients on Day 7.5 after LH injection. Pregnancy was diagnosed 25 days after embryo transfer. Healthy Bactrian camel calves (n = 4) were born without any particular complications at the time of parturition (e.g. dystocia and neonatal diseases). The present study is the first report of the birth of Bactrian camel calves from dromedary camels, as well as the first report of interspecies embryo transfer in old world camelids.

292 EFFECT OF A SINGLE INJECTION OF FSH DISSOLVED IN POLYETHYLENE GLYCOL ON SUPEROVULATION RESPONSE IN KOREAN NATIVE COWS

Superovulation in bovine is usually achieved after several FSH injection in a time-consuming protocol, and the half-life of FSH is very short. This study was carried out to assess the efficiency of embryo production after induction of superovulation with a single injection of FSH (Folltropin-V, Vetrepharm, Canada) dissolved in polyethylene glycol (PEG, 8000 MW, Sigma-Aldrich Corp., St. Louis, MO, USA) in Korean Native cows. Eighty-eight cows were randomly assigned to one of four groups. In Group 1 (control), cows were intramuscularly treated with twice-daily administration of 50 mg FSH for 4 days. In Group 2 and 3, cows received a single subcutaneous injection of 400 mg and 200 mg FSH dissolved in 30% PEG, respectively. In Group 4, cows received a single subcutaneous injection of 200 mg FSH dissolved in 30% PEG given seven days after CIDR insertion. On days 10 to 12 after observed estrus, superovulation was started. Luteolysis was induced by administration of 25 mg of PGF2α (Pharmacia, Puurs, Belgium) IM 2 days after the start of the FSH injection. Donors were timed-inseminated with frozen–thawed semen at 48 and 60 h after the administration of PGF2α. Embryos were collected by non-surgical methods on 7 days after the second AI. The numbers of corpus lutea (CL), total ova, transferable embryos, and progesterone concentration were analyzed by General Linear Models Procedure with the Statistical Analysis System (SAS; SAS Institute, Inc., Cary, NC, USA) and means were compared with the Duncan’s multiple range test. Data on the pregnancy rate was analyzed by chi-square test. P values &lt;0.05 were considered to be significantly different. The number of CL in Group 2 was significantly (P &lt; 0.05) higher compared to Groups 1, 3 and 4 (18.5 ± 7.5 v. 11.2 ± 5.1, 13.1 ± 5.1 and 13.9 ± 6.4, respectively). However, the number of total ova (7.9 ± 6.0 to 10.4 ± 7.2) and transferable embryos (3.7 ± 3.3 to 4.7 ± 3.9) did not differ among treatment groups. In addition, blood progesterone concentrations at embryo recovery did not differ among all groups (10.0 ± 3.5 ng mL–1 to 15.7 ± 3.2 ng mL–1). No difference was observed among groups in pregnancy rate after transferring the recovered embryos (36.0 to 50.0%). In conclusion, our results show that a single injection of FSH dissolved in polyethylene glycol is an efficient and simple procedure for inducing superovulation in Korean native cows.

Short-term treatment with a controlled internal drug releasing (CIDR) device and FSH to induce fertile estrus and increase prolificacy in anestrous ewes

The objectives were to evaluate, in anestrous ewes, the effectiveness of a CIDR-G device (0.3 g progesterone) administered for 5 d to induce estrus; and FSH (Folltropin; 55 mg NIH-FSH-P1 equivalent) in saline: propylene glycol (1:4) 24 h before insert removal (Day 0), to increase ovulation rate and prolificacy. Ewes of mixed breeding were assigned at random to 3 treatments: control (C; n = 125), 5 d progesterone (P5; n = 257) and 5 of progesterone plus FSH (P5F; n = 271). Intact rams were joined at insert removal and ewes were observed every 24 h for 3 d. On Day 14, the ovulation rates of all ewes detected in estrus in the treated groups were determined using transrectal ultrasonography. Rams were removed on Day 26 to 31. Ewes were examined for pregnancy then, and again 20 to 25 d later to detect ewes that conceived to the second service period. Percentage of ewes marked by rams was higher in progesterone-treated (77%) than in C (20%; P < 0.01), but did not differ between P5 and P5F. The ovulation rate (1.95 ± 0.04) did not differ due to FSH. Conception (68%) and pregnancy (52%) rates were higher in progesterone-treated (P < 0.01) than in C (0%) ewes. Estrous response varied quadratically with time after ram introduction, and the conception rate varied quadratically with the time of observation of onset of estrus. Over two service periods more progesterone-treated than C ewes lambed (65 vs 45%; P < 0.01). Lambs born per ewe exposed (0.7 ± 0.1, 1.0 ± 0.1, and 1.1 ± 0.1 for C, P5 and P5F, respectively) was increased by progesterone (P < 0.05). Litter size to the first service period (1.59 ± 0.04) and overall (1.54 ± 0.03) did not differ among treatment groups. FSH-treated ewes tended to have more lambs (1.67 ± 0.1) than did ewes receiving progesterone alone (1.5 ± 0.1; P = 0.06) and than did ewes lambing to the second service period (1.5 ± 0.1; P = 0.06). In summary, a 5-d progesterone pre-treatment of anestrous ewes induced estrous cycles and increased the pregnancy rates. A single injection of FSH only tended to increase litter size.

Superovulation of beef cows and heifers with a single injection of FSH diluted in polyvinylpyrrolidone

Superovulation of beef cows and heifers with a single injection of FSH diluted in polyvinylpyrrolidone

Follicle stimulating hormone stimulates the production of gonadotrophin surge attenuating factor (GnSAF) in vivo

To study the time-course production of gonadotrophin surge attenuating factor (GnSAF) after the onset of FSH treatment in women. Normally cycling women were treated with FSH injections (225 IU per day) starting on cycle day 2 (0800 h). The response of LH to an i.v. injection of 10 micrograms GnRH (GnSAF bioactivity) was investigated 12, 24, 36 and 48 hours after the first FSH injection, as well as during the early follicular phase of an untreated spontaneous cycle. Six normally ovulating women with long-standing unexplained infertility were studied. The women were used as their own controls during the spontaneous cycles. Pituitary response to GnRH was calculated as the net increase in LH at 30 min (delta LH30) above the basal value. delta LH30 was significantly attenuated 12, 24, 36 and 48 hours after the first FSH injection as compared to the spontaneous cycles. In the latter cycles, delta LH30 decreased significantly from day 2 (12 hours) to day 4 (48 hours). Serum oestradiol levels at 12 and 24 hours did not differ significantly between the FSH and the spontaneous cycles. These results demonstrate that in superovulated women, a marked attenuation in the pituitary response to GnRH occurs as early as 12 hours from a single injection of FSH before any significant increase in serum oestradiol levels. It is suggested that FSH is a potent stimulus of GnSAF production in women.

The effect of a single injection of FSH on day 1 of the cycle on the number of follicles and follicular fluid concentrations of estrogen, progesterone and inhibin in heifers

The effect of a single injection of FSH on day 1 of the cycle on the number of follicles and follicular fluid concentrations of estrogen, progesterone and inhibin in heifers

Induction of multiple ovulation in the cow with single injections of FSH and HCG.

A gonadotrophin regimen was developed to induce a high incidence of double and triple ovulations in the beef cow using a single injection of FSH on Day 16 followed by a low dose of HCG on the day of oestrus. Conception rate was not affected by gonadotrophin treatment and 1/9 cows experiencing multiple ovulations gave birth to twin calves.