In vitro and in vivo embryo production in cattle superstimulated with FSH for 7 days

Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles<5 mm decreased during superstimulation suggesting that there was no continuous recruitment of small follicles, and the number of follicles>1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment. (AU)

In vitro and in vivo embryo production in cattle superstimulated with FSH for 7 days

Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment.

Evaluation and classification of bovine embryos

Bovine Embryo transfer has been used widely to reproduce the most valuable females in the herd with about 750,000 embryos produced annually from superovulated donors and more than 450,000 embryos produced are the safest and most cost affective alternatives to move genetics internationally because og their low risk of transmitting diseases. One of most important factors associated with the success and widepread application of this technologie is evaluation of the embryos before freezing and/or transfer to a recipient. Embryos are usually classified based on a number code system for their stage of development (1 to 9) and for their quality (1 to 4). The basic principles of embryo evaluation are briefly described.(AU)

Innovative strategies for superovulation in cattle

Although superstimulatory protocols in cattle are usually initiated during mid-cycle, the elective control follicular wave emergence and ovulation have had a great impact on the application of on-farm embryo transfer. However, the most commonly used approach for the sinchronization of follicular wave emergence invoolves the use of estradiol which cannot be used in many parts of the world. Therefore, the need for alternative treatments has driven recent research. An approach that has shown promise is to initiate FSH treatments at the time of the emergence of the first follicular wave folliwing GnRH-induced ovulation. Alternatively, it has been shown that that it may be possible to ignore follicular wave status, and by extending the treatment protocol induce smaller follicles to grow and reach maturity and superovulate. Finally , the short half-life of pintuitary FSH necessitates twice daily treatments which are times consuming, stressful and subject to error. Recent treatment protocols have permitted supertimulation with a single or alternatively two-FSH treatments, reducing the need for animal handling during FSH treatments.(AU)

History and perspectives on bovine embryo transfer

On a worldwide basis, more than 750,000 embryos are produced annually from superovulated donors and more than 450,000 embryos are produced using in vitro techniques. Superovulation and embryo collection are done as frequentlu as every 30 days. Cryopreservation and direct transfer of frozen-thawed embryos results in pregnancy rates near that of fresh embryos. Since the zona pellucida-infact in vivo produced bovine embryo can be made specified pathogen-free by washing procedures, thousands of frozen embryos are marketed internationally on an annual basis. In vitro embryo production is used widely in countries like Brazil and Japan. Polymerase chain reaction (PCR) technology is currentlu being used for sexing embryos and this technology is beginning to be used for "embryo diagnostics" and "embryoi genomics". Sex-sorted bovine semen is also readily available and is being used increasingly, especially for in vitro embryo production.(AU)

History and perspectives on bovine embryo transfer

On a worldwide basis, more than 750,000 embryos are produced annually from superovulated donors and more than 450,000 embryos are produced using in vitro techniques. Superovulation and embryo collection are done as frequentlu as every 30 days. Cryopreservation and direct transfer of frozen-thawed embryos results in pregnancy rates near that of fresh embryos. Since the zona pellucida-infact in vivo produced bovine embryo can be made specified pathogen-free by washing procedures, thousands of frozen embryos are marketed internationally on an annual basis. In vitro embryo production is used widely in countries like Brazil and Japan. Polymerase chain reaction (PCR) technology is currentlu being used for sexing embryos and this technology is beginning to be used for "embryo diagnostics" and "embryoi genomics". Sex-sorted bovine semen is also readily available and is being used increasingly, especially for in vitro embryo production.

Innovative strategies for superovulation in cattle

Although superstimulatory protocols in cattle are usually initiated during mid-cycle, the elective control follicular wave emergence and ovulation have had a great impact on the application of on-farm embryo transfer. However, the most commonly used approach for the sinchronization of follicular wave emergence invoolves the use of estradiol which cannot be used in many parts of the world. Therefore, the need for alternative treatments has driven recent research. An approach that has shown promise is to initiate FSH treatments at the time of the emergence of the first follicular wave folliwing GnRH-induced ovulation. Alternatively, it has been shown that that it may be possible to ignore follicular wave status, and by extending the treatment protocol induce smaller follicles to grow and reach maturity and superovulate. Finally , the short half-life of pintuitary FSH necessitates twice daily treatments which are times consuming, stressful and subject to error. Recent treatment protocols have permitted supertimulation with a single or alternatively two-FSH treatments, reducing the need for animal handling during FSH treatments.

Evaluation and classification of bovine embryos

Bovine Embryo transfer has been used widely to reproduce the most valuable females in the herd with about 750,000 embryos produced annually from superovulated donors and more than 450,000 embryos produced are the safest and most cost affective alternatives to move genetics internationally because og their low risk of transmitting diseases. One of most important factors associated with the success and widepread application of this technologie is evaluation of the embryos before freezing and/or transfer to a recipient. Embryos are usually classified based on a number code system for their stage of development (1 to 9) and for their quality (1 to 4). The basic principles of embryo evaluation are briefly described.

Prostaglandin F2α promotes ovulation in prepubertal heifers.

The objective was to determine the effects of exogenous prostaglandin F(2α) (PGF), with or without progesterone treatment, on first ovulation in prepubertal heifers. We tested the hypothesis that PGF has a luteolysis-independent ovulatory effect in cattle. Crossbred Angus heifers (12 to 14 mo old, 250 kg body weight, and an average body condition score of 3 out of 5) were examined by transrectal ultrasonography on two occasions, 11 days apart. Heifers in which a CL was not detected at either examination were considered prepubertal. Heifers were assigned randomly to three experimental groups: (1) PG group (N = 14); heifers were treated with a PGF analog (500 µg cloprostenol im) 5 days after the emergence of a spontaneous (i.e., naturally occurring, noninduced) follicular wave; (2) PPG group (N = 12); heifers were given an intravaginal progesterone-releasing insert (CIDR; Pfizer Animal Health, Montreal, QC, Canada), and a follicular wave was induced with 50 mg of progesterone + 2 mg of estradiol benzoate im, and a PGF analog was given at the time of CIDR removal, on day 5 of the follicular wave (on average, 8.6 ± 0.5 days after CIDR insertion); and (3) control group heifers were given no treatment (N = 14). Heifers were examined daily by transrectal ultrasonography from the start of the experiment to confirmation that ovulation had occurred, or to 5 days after PGF injection (PG and PPG groups) or until dominant follicles of the next follicular wave reached 8 mm (control group). The percentage of heifers that ovulated within 10 days after wave emergence was higher in PPG (10/12; 83.3%) and PG (11/14; 78.5%) groups than in control (1/14; 7.1%; P < 0.0001). Ovulations occurred 69.6 ± 6 h and 93.8 ± 5 h after PGF treatment in PPG and in PG groups, respectively, whereas only one heifer in the control group ovulated 96 h after day 5 of follicular wave (P = 0.13). In summary, PGF treatment was associated with ovulation in prepubertal heifers whether or not exogenous progesterone was used as a pretreatment. The hypothesis that PGF will induce ovulation by a luteolysis-independent mechanism was supported.

The effect of timing of the induction of ovulation on embryo production in superstimulated lactating Holstein cows undergoing fixed-time artificial insemination.

Two experiments evaluated the effects of timing of the induction of ovulation in superstimulated lactating Holstein donor cows that were fixed-time artificially inseminated. Secondary objectives were to evaluate the effects of the timing of progesterone (P4) device removal (Experiment 1) or the addition of a second norgestomet implant (Experiment 2) during superstimulation. In Experiment 1, 12 cows were allocated to one of four treatment groups with the timing of P4 device removal (24 or 36 h) and pLH treatment (48 or 60 h), after the first PGF as main factors, in a Latin Square (cross-over) design. There was an interaction (P = 0.03) between time of P4 device removal and time of pLH treatment. Mean (± SEM) numbers of transferable embryos were higher when the P4 device was removed at 36 h and pLH was administered at 60 h after the first PGF (P36LH60 =6.3 ± 1.4) compared to other treatments (P24LH60 =3.7 ± 1.1; P24LH48 =2.4 ± 0.8; or P36LH48 =2.2 ± 0.7). In Experiment 2, 40 cows were randomly allocated into one of four treatments with the number of norgestomet implants (one or two) and the time of induction of ovulation with GnRH relative to the first PGF (48 vs. 60 h) as main effects. The mean number of transferable embryos was higher (P = 0.02) when GnRH was administered at 60 h (4.2 ± 1.3) compared to at 48 h (2.7 ± 0.8), and the number of freezable embryos was increased (P = 0.01) in cows receiving two (3.0 ± 1.0) rather than one norgestomet implant (1.5 ± 0.5). In summary, embryo production in lactating Holstein cows was increased when the ovulatory stimulus (pLH or GnRH) was given 60 h after the first PGF, particularly when the P4 device was removed 36 h after the first PGF and when two norgestomet ear implants were used during the superstimulation protocol.

Increasing pregnancies following synchronization of bovine recipients

The success of a commercial embryo transfer program depends on the production of high numbers of viable embryos from donor cows that result in high numbers of calves born when the embryos are transferred to suitable recipien ts. In recent years, a great deal of effort has been devoted to the development of treatment protocols that permit the efficient use of recipients and result in high pregnancy per recipient synchronized, especially in recipients managed on pasture. One of the most successful alternatives to increase the number of recipients utilized in embryo transfer programs is the use of protocols that allow for embryo transfer without the need for estrus detection, usually called fixed-time embryo transfer (FTET). Pregnancies to FTET have been reported to be similar to those after detection of estrus, but the overall proportion of recipients pregnant over those synchronized are higher because these treatments have increased the proportion of recipients that receive embryos. Treatments that increase pr ogesterone concentrations and pregnancies per embryo transfer have also been investigated in recent years. Most of these treatments have resulted in increased pregnancy per embryo transfer in recipients with Bos indicus influence, while benefit were not so obvious in Bos taurus recipients managed under more optimal conditions. While factors such as the reproductive histor y of the recipients and the stage and quality of embryos may affect pregnancy per embryo transfer, other factors such as estrus detection and the time interval from thawing to transfer do not seem to affect the proportion of recipients pregnant with embryos frozen in ethylene glycol and transferred at a fixed-time.(AU)

Increasing pregnancies following synchronization of bovine recipients

The success of a commercial embryo transfer program depends on the production of high numbers of viable embryos from donor cows that result in high numbers of calves born when the embryos are transferred to suitable recipien ts. In recent years, a great deal of effort has been devoted to the development of treatment protocols that permit the efficient use of recipients and result in high pregnancy per recipient synchronized, especially in recipients managed on pasture. One of the most successful alternatives to increase the number of recipients utilized in embryo transfer programs is the use of protocols that allow for embryo transfer without the need for estrus detection, usually called fixed-time embryo transfer (FTET). Pregnancies to FTET have been reported to be similar to those after detection of estrus, but the overall proportion of recipients pregnant over those synchronized are higher because these treatments have increased the proportion of recipients that receive embryos. Treatments that increase pr ogesterone concentrations and pregnancies per embryo transfer have also been investigated in recent years. Most of these treatments have resulted in increased pregnancy per embryo transfer in recipients with Bos indicus influence, while benefit were not so obvious in Bos taurus recipients managed under more optimal conditions. While factors such as the reproductive histor y of the recipients and the stage and quality of embryos may affect pregnancy per embryo transfer, other factors such as estrus detection and the time interval from thawing to transfer do not seem to affect the proportion of recipients pregnant with embryos frozen in ethylene glycol and transferred at a fixed-time.

Ultrasonographic and endocrine aspects of follicle deviation, and acquisition of ovulatory capacity in buffalo (Bubalus bubalis) heifers.

The objectives of this study were to determine the interval from ovulation to deviation and the diameter of the dominant (DF) and largest subordinate (SF) follicles at deviation in buffalo (Bubalus bubalis) heifers. Two methods of evaluation (observed vs. calculated) were used. FSH and LH profiles encompassing follicle deviation (Experiment 1), and the follicular diameter when the DF acquired ovulatory capacity (Experiment 2) were also determined. The time of deviation and the diameter of the DF and the largest SF at deviation did not differ between observed and calculated methods. Overall, follicle deviation occurred 2.6 ± 0.2d (mean ± SEM) after ovulation, and the diameters of the DF and SF at deviation were 7.2 ± 0.2 and 6.4 ± 0.2mm, respectively. No changes in plasma levels of FSH or LH were observed (P=0.32 and P=0.96, respectively). Experiment 2 was conducted in two phases according to the diameter of the DF during the first wave of follicular development at the time of LH challenge (25mg of pLH). In the first phase, follicles ranging from 5.0 to 6.0mm (n=7), 6.1 to 7.0mm (n=11), or 7.1 to 8.0mm (n=9) were used, and in the second phase, follicles ranging from 7.0 to 8.4mm (n=10), 8.5 to 10.0mm (n=10), or 10.1 to 12.0mm (n=9) of diameter were used. After the pLH treatment, the DF was monitored by ultrasonography every 12h for 48h. No ovulations occurred in heifers in the first phase. However, in the second phase, an effect of follicular diameter was observed on ovulation rate [7.0-8.4mm (0.0%, 0/10), 8.5-10.0mm (50.0%, 5/10), and 10.0-12.0mm (55.6%, 5/9)]. In summary, follicle deviation occurred 2.6d after ovulation in buffalo (B. bubalis) heifers, when the diameters of the DF and SF were 7.2 and 6.4mm, respectively. No significant changes in plasma concentrations of FSH or LH were detected. Finally, the acquisition of ovulatory capacity occurred when the DF reached 8.5mm in diameter.

Treatments for the synchronisation of bovine recipients for fixed-time embryo transfer and improvement of pregnancy rates.

Although embryo transfer technology has been used commercially in cattle for many years, the inefficiency of oestrus detection, especially in recipients, has limited the widespread application of this technology. The most useful alternative to increase the number of recipients utilised in an embryo transfer program is the use of protocols that allow for embryo transfer without the need for oestrus detection, usually called fixed-time embryo transfer (FTET). Most current FTET protocols are based on progestin-releasing devices combined with oestradiol or GnRH, which control and synchronise follicular wave dynamics and ovulation. Conception rates to a single FTET have been reported to be similar to those after detection of oestrus, but pregnancy rates are higher because these treatments have increased the proportion of recipients that receive an embryo. Recent changes to treatments for FTET, such as the administration of eCG, have resulted in increased pregnancy rates and provide opportunities to make these treatments easier to perform on farm.

Control of ovarian function for assisted reproductive technologies in cattle

Although the discovery that follicles in bovine ovaries grow in a wave-like fashion has presented challenges for estrus synchronization and superovulation, recent protocols, designed to control follicular function have permitted fixed-time AI or fixed-time embryo transfer with high pregnancy rates, and the initiation of superstimulatory treatments at a self-appointed time, all without the necessity of estrus detection. The key is the synchronization of follicle wave emergence. More recent studies have revealed that it is not only possible to synchronize the timing of ovulation for fixed-time AI in single ovulating animals, but also in superstimulated donors. Ultrasound-guided follicle ablation is very efficacious in synchronizing follicle wave emergence but is difficult to apply in the field. Similarly, the induction of ovulation with GnRH or LH will effectively synchronize follicle wave emergence, but ovulation occurs in only 60 to 70% of cases. The administration of estradiol benzoate in progestin-treated cattle effectively synchronizes follicle wave emergence for both superovulation and estrus synchronization, but estradiol benzoate is not available in many countries. The challenge now is to use knowledge of follicle wave dynamics to design alternatives.(AU)

Control of ovarian function for assisted reproductive technologies in cattle

Although the discovery that follicles in bovine ovaries grow in a wave-like fashion has presented challenges for estrus synchronization and superovulation, recent protocols, designed to control follicular function have permitted fixed-time AI or fixed-time embryo transfer with high pregnancy rates, and the initiation of superstimulatory treatments at a self-appointed time, all without the necessity of estrus detection. The key is the synchronization of follicle wave emergence. More recent studies have revealed that it is not only possible to synchronize the timing of ovulation for fixed-time AI in single ovulating animals, but also in superstimulated donors. Ultrasound-guided follicle ablation is very efficacious in synchronizing follicle wave emergence but is difficult to apply in the field. Similarly, the induction of ovulation with GnRH or LH will effectively synchronize follicle wave emergence, but ovulation occurs in only 60 to 70% of cases. The administration of estradiol benzoate in progestin-treated cattle effectively synchronizes follicle wave emergence for both superovulation and estrus synchronization, but estradiol benzoate is not available in many countries. The challenge now is to use knowledge of follicle wave dynamics to design alternatives.

Follicle deviation and ovulatory capacity in Bos indicus heifers.

The objectives of Experiment 1 were to determine the interval from ovulation to deviation, and diameter of the dominant follicle (DF) and largest subordinate follicle (SF) at deviation in Nelore (Bos indicus) heifers by two methods (observed and calculated). Heifers (n = 12) were examined ultrasonographically every 12 h from ovulation (Day 0) to Day 5. The time of deviation and diameter of the DF and largest SF at deviation did not differ (P>0.05) between observed and calculated methods. Overall, deviation occurred 2.5+/-0.2 d (mean +/- S.E.M.) after ovulation, and diameters for DF and largest SF at deviation were 6.2+/-0.2 and 5.9 +/- 0.2 mm, respectively. Experiment 2 was designed to determine the size at which the DF acquires ovulatory capacity in B. indicus heifers. Twenty-nine heifers were monitored every 24 h by ultrasonography, from ovulation until the DF reached diameters of 7.0-8.4 mm (n=9), 8.5-10.0 mm (n=10), or >10.0 mm (n=10). At that time, heifers were treated with 25 mg of pLH and monitored by ultrasonography every 12 h for 48 h. Ovulation occurred in 3 of 9, 8 of 10, and 9 of 10 heifers, respectively (P<0.05). In summary, there was no significant difference between observed and calculated methods of determining the beginning of follicle deviation. Deviation occurred 2.5 d after ovulation when the DF reached 6.2 mm, and ovulatory capacity was acquired by DF as small as 7.0 mm.

The control of follicular wave development for self-appointed embryo transfer programs in cattle.

Our expanding knowledge of the control of follicular wave dynamics during the bovine estrous cycle has resulted in renewed enthusiasm for the prospects of precisely controlling the follicular and luteal dynamics and finely controlling the time of ovulation. Follicular wave development can be controlled mechanically by ultrasound-guided follicle ablation or hormonally by treatments with GnRH or estradiol and progestogen/progesterone in combination. Treatment of cattle with GnRH in combination with prostaglandin F2 alpha (PGF) 7 d later and a second GnRH 48 h after PGF (known as Ovsynch) has resulted in acceptable pregnancy rates after fixed-time AI in lactating dairy cows and in recipients in which embryos were transferred without estrus detection. Alternatively, treatments with estradiol and progestogen/progesterone-releasing devices resulted in synchronous emergence of a new follicular wave and, when a second estradiol treatment was given 24 h after device removal, synchronous ovulation and high pregnancy rates to fixed-time AI. Self-appointed embryo transfer (without estrus detection) using estradiol and progesterone treatments have resulted in pregnancy rates comparable with those obtained with recipients transferred 7 d after estrus. Furthermore, estradiol and progesterone treatments combined with PGF and eCG (given 1 d after the expected time of wave emergence) have resulted in high rates of recipients selected for transfer (84.6%) and an overall pregnancy rate of 48.7% (recipients pregnant/recipients treated). Estradiol and progestogen/progesterone treatments have also been widely used for self-appointed superstimulation protocols with equivalent embryo production to that of donor cows superstimulated using the traditional approach beginning 8 to 12 d after estrus. In summary, exogenous control of luteal and follicular development facilitates the application of assisted reproductive technologies in cattle by offering the possibility of planning the superstimulation of donors and synchronization of recipients at a self-appointed time, without the necessity of estrus detection and without sacrificing results.

Transvaginal collection and ultrastructure of llama (Lama glama) oocytes.

Ultrasound-guided transvaginal follicle aspiration has been described as a noninvasive and repeatable procedure for oocyte collection in several species, but its use has not been described for any of the members of the family, Camelidae. A study was designed to determine the feasibility of an ultrasound-guided transvaginal approach for oocyte collection in llamas. Fifteen non-pregnant, adult female llamas (10 non-stimulated and 5 superstimulated) were examined by transrectal ultrasonography with a 7.5-MHz linear-array transducer to determine the number and diameter of follicles available for aspiration. After caudal epidural anesthesia was induced, the 7.5-MHz linear-array transducer was fastened to a long rigid handle and inserted intravaginally. The free hand was placed into the rectum to manipulate the ovaries, one at a time, in position against the vaginal wall over the face of the transducer. A 20-gauge, 55-cm-long, single-lumen needle was advanced through the vaginal fornix and into follicles > or = 3 mm in diameter. Follicular contents were aspirated using a regulated vacuum pump (flow rate = 33 mL/min; approximately 150 mm Hg) into a tube containing 3 mL of phosphate buffered saline and 0.2% BSA. Fluid was filtered (75 microm mesh), and oocytes were located and morphologically evaluated using a stereomicroscope. Overall, 134 follicles were aspirated, and 76 oocytes were collected (collection rate = 57%). Thirty-two oocytes (42%) were surrounded by multiple layers of compacted granulosa cells and had homogenous dark ooplasm; 13 oocytes (17%) were surrounded by the corona radiata layer only and had heavily granulated ooplasm; 9 oocytes (12%) were denuded and had homogenous dark ooplasm; and 22 oocytes (29%) were denuded and displayed signs of ooplasm degeneration. The ultrastructure of llama oocytes was similar to that of cattle except for conspicuous accumulation of large lipid droplets in the cytoplasm. Twenty-four hours after follicle aspiration, the ovaries were examined by transrectal ultrasonography and intrafollicular hematomas were detected in 3 llamas (9 of 48 follicles aspirated). Results demonstrate the potential utility of a transvaginal ultrasound-guided technique for oocyte collection and in vitro embryo production in llamas. Oocytes of llamas bear an ultrustructural resemblance to those of cattle, but are distinguished by a predominance of cytoplasmic lipid.