Academic literature on the topic 'Superstimulation treatment'

Fertility of lactating dairy cows is associated with reduced progesterone (P4) concentration compared with nonlactating animals. The objective of the current study was to determine whether P4 during growth of the first follicular wave (FFW) affects embryo quality. Lactating Holstein cows at 33±3 days post partum were allocated to one of three treatments. Cows in the FFW and FFW with P4 (FFWP) treatments started the superstimulation protocol on day 1 of the estrous cycle and second follicular wave (SFW) cows started the superstimulation protocol on estrous cycle day 7. Cows were superstimulated with 400 mg of NIH-FSH-P1 (FSH) given twice daily for 5 days, two prostaglandin F2α (PGF2α) injections given with the ninth and tenth injections of FSH, GNRH given 48 h after the first PGF2α injection, and timed insemination 12 and 24 h after the GNRH injection. Cows in the FFWP treatment received two intravaginal P4 inserts during the superstimulation. Embryos were recovered 6.5 days after artificial insemination and excellent/good and fair embryos were frozen and transferred. Blood was sampled daily from estrous cycle day 0 until insemination from donor cows. During the superstimulation protocol, P4 was (P<0.01) greatest for SFW cows followed by FFWP and FFW cows respectively. The percentage of embryos–oocytes from SFW and FFWP cows classified as excellent/good and fair embryos was (P=0.02) greater than those of FFW cows. Pregnancy per embryo transfer was not (P≥0.73) affected by embryo donor treatment. Reduced embryo quality of cows induced to ovulate the follicles from the first follicular wave is a consequence of reduced P4 during follicle growth.

Protocols that control follicular wave emergence and ovulation have had a great impact on the application of commercial on-farm embryo transfer because they permit the initiation of superstimulatory treatments at a self-appointed time. However, the most common approach for the synchronization of follicular wave emergence for superstimulation involves the use of estradiol or its esters that are not commercially available in many countries. Therefore, an experiment was designed to evaluate a protocol in which the superstimulation treatment began at the time of emergence of the first follicular wave without the use of estradiol. Bonsmara donors (29 cows and 41 heifers) were randomly allocated to one of two treatment groups. Donor animals in the experimental group (first wave group) received an intravaginal progesterone releasing device (Cue-Mate, Bioniche Animal Health, ON, Canada) along with PGF (0.150 mg D + cloprostenol, Bioprost-D, Biotay, Argentina) at random stages of the estrous cycle. Cue-Mates were removed 10.5 d later and a second PGF was administered at the same time, followed by GnRH (0.050 mg Lecirelina, Biosin-OV, Biotay, Argentina) 36 h later. Ovulation was expected to occur within 30 h after GnRH (day 0). On day 0 (36 h after gonadotropin-releasing hormone) donors received a new Cue-Mate, and superstimulation treatment was initiated with a total dose of 200 to 260 mg (heifers) or 320 mg (cows) NIH-FSH-P1of Folltropin-V in twice daily decreasing doses over 5 d. The PGF was administered with the last two Folltropin-V injections, and Cue-Mate devices were removed with the last Folltropin-V injection. All donors received 12.5 mg pLH (Lutropin-V, Bioniche Animal Health) 24 h after Cue-Mate removal and were AI 12 and 24 h later. Embryos were collected 7 d after pLH treatment. Donors in the Control group received a Cue-Mate and 2 mg of estradiol benzoate (EB; Bioestradiol, Biotay) and 50 mg of progesterone (Lab. Rio de Janeiro, Argentina), and superstimulation treatments were initiated 4 d later with the same dosages used in the first wave group. The PGF administration, Cue-Mate removal, AI, and embryo collections were done as those in the first wave group. Data were analyzed by ANOVA, and results are shown in Table 1. It was not possible to pass the cervix with the collection catheter in two heifers in the control group, and they were excluded from the analysis. There were no significant effects of donor category (cows v. heifers) or treatment on superovulatory response and embryo quality (P > 0.20). In conlusion, superstimulation on a synchronized first follicular wave is as efficacious as superstimulation following synchronization of follicle wave emergence with estradiol benzoate in Bonsmara cattle. Table 1. Superovulatory response (means ± SEM) in Bonsmara cows and heifers treated with Folltropin-V during the first follicular wave or 4 d after estradiol administration Bioniche Animal Health, Belleville, ON, Canada.

Although we have previously shown that ovarian superstimulation during the first follicular wave resulted in a successful response (Carballo Guerrero D et al. 2009 Reprod. Fertil. 21, 242), the current protocol needs to be optimized in order to be used in the field. Therefore, an experiment was designed to simplify this treatment and to compare it with the traditional superstimulation protocol using progesterone and estradiol. Simmental cows (n = 14) were subjected to 3 superstimulation treatments (2 first wave groups and 1 control group) in a crossover design (i.e. all cows received the 3 treatments and all treatments were represented on each collection day). Cows in Group 1 received a progesterone-releasing device (Cue-Mate®, Bioniche Animal Health, Belleville, Ontario, Canada) along with 0.150 mg of D + cloprostenol (PGF; Bioprost-D®, Biotay, Buenos Aires, Argentina) at random stages of the estrous cycle. A second PGF was injected 5 days after Cue-Mate® insertion, followed by GnRH (0.050 mg of lecirelin; Biosin-OV®, Biotay) 36 h later (i.e. 7 days after Cue-Mate® insertion). Based on previous studies, ovulation was expected to occur 30 to 36 h later. Therefore, superstimulation treatments were initiated 36 h after GnRH (Day 0), with a total dose of 400 mg NIH-FSH-P1 of Folltropin®-V (Bioniche Animal Health) in twice-daily decreasing doses over 4 days. Prostaglandin was administered with the last 2 Folltropin®-V injections and Cue-Mate® devices were removed with the last Folltropin®-V injection. Cows received 12.5 mg of porcine LH (Lutropin®-V, Bioniche Animal Health) 24 h after Cue-Mate® removal and were AI 12 and 24 h later. Ova/embryos were collected 7 days after porcine LH and evaluated following IETS recommendations. Cows in Group 2 were treated similarly to those in the Group 1, except they did not receive the second PGF injection 5 days after Cue-Mate® insertion (thus eliminating the need to handle animals on that day). Finally, cows in Group 3 [estradiol benzoate (EB)+P4 control group] received a Cue-Mate® plus 2.5 mg of EB (Bioestradiol®, Biotay) and 50 mg of progesterone (P4; Lab., Rio de Janeiro, Argentina) at random stages of their estrous cycle. Superstimulation treatments were initiated 4 days later (Day 0) following the same protocol used in Group 1. Data were transformed to square root and analyzed by ANOVA. Mean (± SEM) numbers of ova/embryos collected, fertilized ova, and transferable embryos did not differ among groups (12.9 ± 2.0, 9.8 ± 1.7, and 6.6 ± 1.2; 11.5 ± 1.7, 9.3 ± 1.5, and 7.7 ± 1.6; and 14.5 ± 2.8, 9.4 ± 2.3, and 6.8 ± 1.7 for Groups 1, 2, and 3, respectively). In conclusion, data demonstrated that superstimulation during the first follicular wave can be successfully used in groups of randomly cycling donors without the need for estrus detection or estradiol to synchronize follicular wave emergence. The protocol is easy to follow and embryo production is comparable to that of the estradiol and progesterone protocol.

Although we have previously shown that superstimulation during the first follicular wave resulted in a successful response (Carballo Guerrero D et al. 2007 Reprod. Fertil. Dev. 20, 226), the protocol required many interventions that could influence its application in the field. Therefore, two studies were designed to simplify the superstimulation treatment protocol. Experiment 1 was designed to determine whether it was necessary to remove the progesterone releasing device during the superstimulation protocol. Angus cows (n = 37) were superstimulated by two treatments in a crossover design. Cows in Group 1 (control) received a progesterone releasing device (Cue-Mate, Bioniche Animal Health, Belleville, ON, Canada) along with 0.150 mg D cloprostenol (PGF, Bioprost-D, Biotay, Argentina) IM, at random stages of the estrous cycle. Five days later, a second PGF was injected and Cue-Mates were removed, followed by GnRH (0.050 mg Lecirelina, Biosin-OV, Biotay) 36 h later; ovulation was expected to occur 30 to 36 h later. On Day 0 (36 h after GnRH) donors received a new Cue-Mate and superstimulation treatment was initiated with a total dose of 400 mg NIH-FSH-P1 of Folltropin-V (FSH, Bioniche Animal Health) in twice daily decreasing doses over 5 days. PGF was injected with the last two FSH injections and Cue-Mates were removed with the last FSH injection. Cows in Group 2 were treated similarly to those in the control group, except that Cue-mate devices were not replaced and remained in place for 13 days (i.e. Cue-mates were removed with the last FSH and PGF injection). All donors received 12.5 mg pLH (Lutropin-V, Bioniche Animal Health) 24 h after Cue-Mate removal and were AI 12 and 24 h later. Embryos were collected 7 days after pLH. Means were compared between groups by Student’s t-test. Superovulatory response and embryo production did not differ between groups. Mean (± SEM) number of ova/embryos collected and transferable embryos were 8.2 ± 1.0 and 4.1 ± 0.6 v. 9.8 ± 0.9 and 5.7 ± 0.7 for Groups 1 and 2, respectively (P > 0.2). Experiment 2 was designed to evaluate the effect of giving FSH for 4 v. 5 days. Simmental (n = 18) and Angus (n = 6) cows were superstimulated by the two treatment protocols in a crossover design. Cows in both groups were treated similarly to those in Group 2 in Experiment 1 (i.e. Cue-Mates were not replaced during treatment). Cows in Group 1 (control) received FSH over 5 days (as in Group 2 of Experiment 1); while those in Group 2 received the same dosage of FSH, but given in twice daily decreasing doses over 4 days (Cue-Mates were removed with the last FSH and PGF injections). Superovulatory response and embryo production did not differ among groups. Mean (± SEM) number of ova/embryos collected and transferable embryos were 13.5 ± 2.4 and 6.6 ± 1.1 v. 12.0 ± 1.9 and 5.8 ± 1.0 for Groups 1 and 2, respectively (P > 0.6). In conclusion, superstimulation of cattle at the time of emergence of the first follicular wave after ovulation results in an acceptable superovulatory response and all treatments evaluated were user-friendly and equally efficient.

Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were upregulated. To validate the microarray results, five genes were selected for real-time PCR (NTS, FOS, THBS1, FN1, and IGF2). Expression of four genes increased significantly in the three different animals tested (NTS, FOS, THBS1, and FN1). The upregulated genes are related to matrix remodeling (i.e. tissue proliferation), disturbance of angiogenesis, apoptosis, and oxidative stress response. We conclude that superstimulation treatment i) results in granulosa cells that lag behind in maturation and differentiation (most of the upregulated genes are markers of the follicular growth stage), ii) activates genes involved with the NFE2L2 oxidative stress response and endoplasmic reticulum stress response, and iii) disturbs angiogenesis.

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the ‘P-36 protocol’ for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT2 receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus–oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.

Effectiveness of in vitro production of embryos (IVP) is limited among other factors by the recovery rate of oocytes. Gonadotropin superstimulation can improve the recovery rate of oocytes. The effect of FSH treatment on follicular population and recovery rate of oocytes at ovum pick-up (OPU) in the growing phase of the 1st as well as the 2nd follicular wave after superstimulation was the object of our experiment. Twelve unpregnant milking cows (15&ndash;20 kg milk per day) housed on a dairy farm were used in the experiment. The cows bearing corpus luteum were synchronized by PGF₂(day 0) and they were treated by FSH (Folicotropin inj. sicc. ad us vet., Spofa Prague, Czech Republic, single doses 80, 80, 80, 80, 40 and 40 UI) in 12 h intervals on days 12, 13 and 14. Transvaginal ultrasonographic puncture of oocytes in cows bearing a new corpus luteum was performed on day 7 (OPU 1, various phase of the follicular wave, removal of the dominant follicle) and it was repeated on days 10 (OPU 2, growing phase of the follicular wave &ndash; control), 16 (OPU&nbsp;3, growing phase of the first follicular wave after superstimulation) and 20 (OPU 4, growing phase of the second follicular wave after superstimulation). All follicles &gt; 2 mm were punctured. The ovarian follicles (ultrasonographically) and numbers and qualities of obtained oocytes (microscopically) were evaluated during and immediately after OPU. Follicular population was divided to small (FS, 2&ndash;5 mm), medium (FM, 5&ndash;9 mm) and large (FL, &gt; 9 mm) follicles. Oocytes were classified as 1st (intact cumulus, &gt; 3 layers of cumulus cells), 2nd (complete 1&ndash;3 layers of cumulus cells), 3rd (incomplete layers of cumulus cells, expanded cumulus mass) and 4th (absence of corona cells, degenerated oocytes) classes. Although we found the least of FS (x = 1.0) during OPU 3, significantly more FM (x = 24.7) and FL (x = 3.1) follicles were found at this procedure in comparison with others. Likewise a significantly higher number of oocytes (x = 8.1) was obtained at OPU 3 in comparison with OPU 1 and OPU 2. Significantly higher number of FM (x = 6.1) was found and non-significantly higher number of oocytes was obtained at OPU 4 in comparison with OPU 1 and 2. The results show that administration of FSH increases the number of follicles and the number of collected oocytes in the growing phase of the 1st follicular wave after superstimulation, nevertheless a higher number of follicles and a higher recovery rate of oocytes can be expected in the growing phase of the 2nd follicular wave after superstimulation as well.

In vitro embryo production (IVP) is an important tool to enhance genetic gain in cattle. However, oocyte quality is a limiting factor for the success of IVP programs in high-producing donors. A series of studies using protocols for follicular wave synchronization and superstimulation before ovum pickup were performed to improve the efficiency of ovum pickup and in vitro production in dairy cattle. The first study evaluated superstimulation with FSH (Folltropin-V®) before ovum pickup in lactating (n = 15) and non-lactating (n = 15) Holstein donors in a crossover design. Cows underwent synchronization of follicle wave emergence (FWE) and at the expected time of FWE, the FSH group received a total dosage of 200 mg of FSH in 4 decreasing doses 12 h apart; controls received no FSH, and ovum pickup was conducted 72 h after the expected FWE in all cows. The FSH-treated cows had a higher (P < 0.01) percentage of medium-sized follicles (6 to 10 mm) at the time of ovum pickup (55.1%) than control cows (20.8%) as well as lower cumulus‐oocyte complexes (COC) recovery rates (60.0 v. 69.8%, respectively; P = 0.002). However, FSH-treated cows had a higher blastocyst production rate (34.5 v. 19.8%; P < 0.01) and more transferable embryos per ovum pickup session (3.0 ± 0.5 v. 1.8 ± 0.4; P = 0.02). Subsequent trials evaluated plasma FSH profiles in 23 heifers and in vitro production following ovum pickup in 90 non-lactating Holstein donors superstimulated with a single IM injection of FSH in 0.5% hyaluronan (HA; MAP-5®, 50 mg). Controls received no treatment, while the F200 group received 200 mg of FSH in 4 decreasing doses 12 h apart. The F200HA and F300HA groups received 200 or 300 mg of FSH in 5 or 7.5 mL, respectively, of 0.5% HA by a single IM injection. Circulating FSH area under curve (AUC) in FSH-treated animals was greater than in the control group (P = 0.02). Although the AUC for F200 group did not differ from HA groups (P = 0.56), the total period of time plasma FSH levels were elevated was greater than in the HA groups (P < 0.01). In the IVP trial, FSH-treated cows had a greater proportion of medium-sized (6–10 mm) follicles than controls (P < 0.001). Also, numbers of follicles (P = 0.01) retrieved (control: 13.1 ± 1.0; F200: 16.5 ± 1.2; F200HA: 19.5 ± 2.1; F300HA: 15.4 ± 1.4; P = 0.01) and blastocysts produced per ovum pickup session (control: 2.4 ± 0.5; F200: 3.7 ± 0.7; F200HA: 4.7 ± 0.7; F300HA: 3.1 ± 0.6; P = 0.06) were greater in cows receiving FSH, regardless of treatment. Cows in the F200HA group had a greater recovery rate (P = 0.009), number of COC cultured (P = 0.04), and blastocysts per ovum pickup session (P = 0.06) than cows in the F300HA group. In conclusion, superstimulation of Holstein donors before ovum pickup increased the efficiency of in vitro production. Additionally, a single IM dose of FSH in 0.5% HA resulted in similar plasma FSH profiles to twice-daily FSH treatment. Non-lactating donors treated with FSH produced more embryos per ovum pickup session regardless of FSH treatment. Lastly, all in vitro-produced endpoints were greater following a single dose of 200 mg of FSH in 0.5% HA than 300 mg of FSH in 0.5% HA.

Genetic improvement in cattle has focused in recent years on the large reproductive potential that resides in the ovaries of females at an early age. It is estimated that approximately 150,000 oocytes are present in primordial follicles in foetal ovaries at birth, and recruitment of follicles from the primordial pool has been initiated by the time of birth. Further, follicular growth can be superstimulated in heifer calves from around 4 weeks of age by treatment with gonadotrophins, and oocytes recovered and placed through in vitro maturation and fertilisation procedures to produce viable embryos.

The capacity to use embryos derived from heifer calves has the potential to reduce generation intervals and increase the rates of genetic gain in cattle. Studies on embryo production from heifer calves have reported inconsistent and unpredictable responses to superstimulation of follicle growth with FSH, similar to that observed in sexually mature heifers. Heifer calves that had a relatively large (>10mm) follicle on the ovary at the end of superstimulation, had a smaller number of total follicles compared with heifer calves that did not have a large follicle on the surface of the ovary. This observation led to the suggestion that follicular interrelationships may occur from an early age in heifers, and that a large follicle may suppress the development of other follicles. Nutrition appears to influence ovarian follicle status in peri-pubertal and pubertal heifers and possibly the response to superstimulation of follicular growth in older animals. There may be a role, therefore, for nutrition in ovarian follicle growth and responses to superstimulation in heifer calves.

In a number of studies oocytes obtained from heifer calves were reported to have a reduced developmental competency in vitro compared with oocytes obtained from ovaries of post-pubertal heifers.

In cattle, treatment with agonists of gonadotrophin hormone releasing hormone (GnRH) desensitises the anterior pituitary gland to GnRH which blocks pulsatile secretion of LH but allows continued basal LH secretion. Antagonists of GnRH prevent both pulsatile and basal secretion of LH. It is possible that treatment with GnRH agonists and antagonists might be used to regulate gonadotrophin secretion in heifer calves and prevent the development of large (functionally dominant) follicles. Subsequent initiation of superstimulation when a pool of small gonadotrophinresponsive follicles are present on the ovaries, and maturing these follicles in synchrony in vivo, may allow a pool of oocytes at similar stages of maturation to be collected for in vitro procedures.

The first two experiments in this thesis examined the requirement of LH for oocyte maturation by treating calves with gonadotrophin hormone releasing hormone (GnRH) agonist and antagonist before and during superstimulation with FSH. Simultaneous treatment with a GnRH agonist during superstimulation of ovarian follicle growth with FSH tended to increase the number of follicles stimulated to grow and significantly increased the number of Grade A and Grade B oocytes collected. In a second experiment, treatment with a GnRH antagonist tended to increase blastocyst development rate after in vitro fertilisation. It was concluded from these findings that exposure of oocytes to pulsatile secretion of LH, and/or a 'pre-ovulatory like' surge release of LH, is not an obligatory requirement for oocyte growth and development in heifer calves.

A third experiment examined the effects of nutrition and growth rate on maturation of the reproductive endocrine axis and the response of calves to superstimulation of ovarian follicle growth with FSH. Heifer calves were raised on two planes of nutrition (relatively low and high) and subsequently superstimulated with FSH. The nutritional treatments resulted in a significant difference in growth rate between the two groups of heifers. However, there were no apparent differences in ovarian follicular responses to stimulation with FSH, oocytes recovered, or in vitro developmental competency of oocytes, between the two groups of heifers.

In the fourth experiment, in vitro developmental competency was compared between oocytes obtained from heifer calves superstimulated with FSH, heifer calves that had not undergone superstimulation and post-pubertal heifers and cows that had not been stimulated with FSH. There were no differences in developmental competency between Grade A and Grade B oocytes derived from the three groups of animals. This finding demonstrated that oocytes obtained from pre -pubertal heifers do not have an intrinsic reduced capacity for in vitro development compared with oocytes obtained from post -pubertal heifers.

The ultimate test of viability of embryos derived from heifer calves is the transfer to recipients and the birth of calves. The aim of the fifth experiment therefore was to test the viability of embryos derived from 10 week-old heifer calves in which ovarian follicular growth was superstimulated with FSH. Transfer of blastocysts produced from oocytes obtained from heifer calves to recipient sexually mature heifers resulted in the birth of normal calves.

In summary, the competency of oocytes collected from heifer calves from an early age has been well established in the series of experiments undertaken in this thesis. The use of superstimulation protocols with heifer calves pre-treated with a GnRH agonist or antagonists increased the number of Grade A and Grade B oocytes, and tended to increase the development to blastocyst post-fertilisation. Grade A and Grade B oocytes collected from heifer calves form blastocysts at rates comparable to oocytes collected from mature cows, and establish pregnancies which result in the birth of calves.