Does synchronising follicular wave emergence improve outcomes in non‐superstimulated donor cattle undergoing ovum pick‐up (OPU) for in vitro embryo production?

Abstract

In vitro embryo production (IVEP) is now a widely commercialised assisted reproductive technology used in cattle reproduction. The use of follicle‐stimulating hormone (FSH) to superstimulate donor cattle prior to ovum pick‐up (OPU) is generally regarded as an effective strategy to improve oocyte quality, developmental competence and increase embryo development rates. However, its use is expensive, labour‐intensive and subject to supply shortages and may attract negative societal perceptions of hormonal stimulation. An alternative strategy involves manipulating normal ovarian physiology through synchronising follicular wave emergence (FWE), thereby generating a homogeneous cohort of common growth phase follicles for oocyte recovery at OPU without the need for FSH. This multi‐PICO critically appraised topic (CAT) examined the effectiveness of synchronising FWE as an alternative to FSH‐dependent programmes in cattle IVEP. A total of 345 studies were identified, nine of which met the inclusion criteria for this study. Only two studies investigated non‐oestrogen synchronisation methods, whereas seven investigated oestrogen‐based protocols. The findings from the seven suitable experimental studies suggest there is currently equivocal evidence for the use of oestrogen‐based synchronisation protocols prior to OPU. The Jadad grading scale adopted for appraisal found the included studies to be insufficient in providing overall certainty in results. Further research into alternative techniques, as well as alterations in study design and population sizes included, would be beneficial, as due to a lack of available evidence, no clear conclusions can be drawn.

In vitro embryo production (IVEP) is increasing in both the dairy and beef industries worldwide, with extensive uptake in North America, South America, Europe and Australasia. ¹ This is owed to its application on a wide range of animals (cows, heifers and juvenile animals), ² , ³ of varying reproductive status (nonpregnant or first‐trimester pregnant), ⁴ and more efficient usage of semen, including reverse‐sorted sperm. ⁵ According to the most recent data reported by the International Embryo Technology Society (IETS), more than 2 million bovine embryos were produced worldwide in 2024, of which in vitro‐derived embryos accounted for more than 80% of the total production, compared to in vivo‐derived embryos produced through Multiple Ovulation Embryo Transfer (MOET). ¹ Despite the global adoption of IVEP, the process is still arguably inconsistent, and overall pregnancy rates often remain lower than other breeding methods, such as artificial insemination and in vivo‐derived/MOET embryos. ⁶ , ⁷ This disparity is even further pronounced in cryopreserved IVP embryos. ⁸

The overall success of IVEP is contingent on the number of high‐quality oocytes recovered during OPU that are capable of completing in vitro maturation (IVM), undergoing fertilisation and progressing through subsequent in vitro culture as an embryo. ⁹ Therefore, outcomes are largely governed by the proportion of recovered oocytes possessing high developmental potential. The simple definition of developmental competence relates to the ability of an oocyte to resume meiosis, cleave following fertilisation and develop into a blastocyst. ¹⁰ However, it may also extend into the formative oocyte's ability to produce a pregnancy that is maintained to term in good health and development, resulting in the delivery of live and vigorous offspring. ¹¹ This highlights opportunities to investigate strategies that enhance both the number and developmental competence of oocytes available via OPU through targeted modulation of ovarian physiology.

It has been shown that follicles in an advanced state of atresia house oocytes with lower developmental competence. ¹² , ¹³ , ¹⁴ Therefore, many IVEP protocols, particularly in North America and Europe, ¹ will administer exogenous follicle‐stimulating hormone (FSH) to superstimulate prospective donor cattle and “rescue” subordinate follicles from atresia. ¹⁵ , ¹⁶ Considerable evidence suggests that follicle size and oocyte competence are improved through superstimulation with FSH. ¹⁷ , ¹⁸ Despite positive outcomes, superstimulatory protocols are labour‐intensive; generally requiring multiple injections in 12‐h intervals over several days, ¹⁹ incurring a high cost; are subject to supply shortages; and carry the potential for negative societal perceptions of hormonal stimulation. This suggests the need for innovation within IVEP systems to find cost‐effective alternatives. Modulation of ovarian follicular dynamics through synchronisation protocols that capture a homogeneous cohort of follicles during the common growth, or early dominant phase, may represent an alternative strategy to improve OPU efficiency without the need for FSH stimulation. ²⁰ This study, therefore, aims to critically appraise the available evidence on synchronising FWE and its effect on the number, quality and developmental competence of oocytes in non‐superstimulated donor cattle.

Clinical scenario

You are a veterinarian providing advanced reproduction services to a Holstein‐Friesian dairy farm in southern Australia that regularly presents high‐value donor cattle for OPU. In the past, there has been a shortage of FSH, and due to high costs, the farmer asks you whether FSH is necessary in the protocol or if there are suitable alternatives. You are aware of oestrous synchronisation protocols and wonder if synchronising FWE prior to OPU could provide a suitable alternative to superstimulation by maximising oocyte yield and quality without the expense of FSH.

Question

In non‐superstimulated cattle undergoing ovum pick‐up and IVEP does synchronising follicular wave emergence (FWE) result in increased number, quality and developmental competenc of oocytes?

Search strategy and outcome

This multi‐PICO study investigated the number, quality and developmental competence of oocytes following synchronisation of non‐superstimulated cattle undergoing OPU‐IVEP. The search strategy was conducted on August 9th, 2025. It included the use of three electronic databases available through the University of Sydney library: CABI: CAB Abstracts (via Web of Science), Scopus (via Elsevier) and ProQuest. Studies published within the last 20 years were included. Identical search terms were used for each database. The terms relating to “bovine” and “cattle” are inclusive of both Bos indicus and Bos taurus species. The term “synchronisation” was used to capture a synchronisation protocol that is applicable to this clinical scenario. Several synchronisation protocols were found to exist in the literature and included in the critical appraisal. The search strategy in ProQuest used “Abstract and Summary”, CAB Abstracts used “Topic” and Scopus used “All fields”. The search terms are listed in Table 1 and the inclusion and exclusion criteria are shown in Table 2. A total of 345 studies were identified, with nine studies meeting the inclusion criteria for this study after removal of duplicates. The results of the search strategy are shown in Table 3. The descriptive data, main findings and limitations are outlined in Table 4, and a summary of the results is presented in Table 5.

Table 1.

Search strategy used in electronic databases to investigate if synchronising follicular wave emergence improves OPU–IVEP in nonstimulated cattle

Databases searched and dates covered	CABI: CAB abstracts (via web of science): 2005 – Present
	Scopus (via Elsevier): 2005 – Present
	ProQuest: 2005 – Present
Dates of searches performed	23rd May 2025, 9th August 2025
Search strategy	CABI: CAB abstracts: TS = (cattle OR bovine OR heifers OR cows) AND TS = (follicular AND wave AND synchronisation OR follicular AND wave AND synchronization OR follicular AND wave AND emergence OR follicular synchronisation OR follicular synchronization OR follicular dynamics) AND TS = (in AND vitro AND embryo AND production OR in vitro fertilization OR in AND vitro AND fertilisation OR IVF OR IVP OR IVEP) AND TS = (OPU OR ovum pick up OR ovum pick‐up) Scopus: ALL (cattle OR bovine OR heifers OR cows) AND ALL (follicular AND wave AND synchronisation OR follicular AND wave AND synchronization OR follicular AND wave AND emergence OR follicular synchronisation OR follicular synchronization OR follicular dynamics) AND ALL (in AND vitro AND embryo AND production OR in AND vitro AND fertilization OR in AND vitro AND fertilisation OR IVF OR IVP OR IVEP) AND ALL (OPU OR ovum pick up OR ovum pick‐up) ProQuest: NOFT (cattle OR bovine OR heifers OR cows) AND NOFT (follicular AND wave AND synchronisation OR follicular AND wave AND synchronization OR follicular AND wave AND emergence OR follicular synchronisation OR follicular synchronization OR follicular dynamics) AND NOFT (in AND vitro AND embryo AND production OR in AND vitro AND fertilization OR in AND vitro AND fertilisation OR IVF OR IVP OR IVEP) AND NOFT (OPU OR ovum pick up OR ovum pick‐up)

Table 2.

Exclusion & inclusion criteria to filter out search results irrelevant to the research question

Exclusion	Published prior to 2005 Use of superstimulatory hormones (FSH, equine chorionic gonadotrophin, human chorionic gonadotrophin, bovine somatotropin) Slaughterhouse OPU Alternative synchronisation modality (photoperiodicity, anti‐Mullerian hormone, nutritional) Controlled trials, conference proceedings, review articles or book chapters
Inclusion	English language Bos taurus and Bos indicus species Randomised controlled trials and crossover studies

Table 3.

Total number of results from the search of three electronic databases

Database	Number of results (English)	Excluded – Pre‐2005	Excluded – Review articles, conference papers, book chapters, thesis	Excluded – Irrelevant to PICO	Total relevant papers
CAB abstracts	37	2	4	26	5
Scopus	278	23	72	179	4
ProQuest	30	4	1	19	6
Total relevant papers after duplicates removed					9

Table 4.

Descriptive analysis, intervention, outcomes, major findings and limitations of the relevant database results

Ref.	Paper	Study information	Intervention and outcomes studied	Main findings	Limitations
21	Anchordoquy et al., 2024	Argentina 2 × 2 crossover study 33 Bos taurus cows (Angus) Cycling cows More than 55 days postpartum BCS between 7 and 5/9 (mean = 6.5) Extensive grazing pasture conditions	Oestrogen COCs recovered (n) Viable COCs (n) Embryos (n)	No significant difference between treatment and control groups in: COC recovery Viable COCs Embryos	No sample size calculations Denuded COCs were included as viable for IVEP Jadad score 2/5
22	Cavalieri et al., 2018	Brazil 2 × 2 crossover study 20 Bos indicus cows (Nelore) Nonpregnant Nonlactating Regular oestrus cycles 3–10 years of age Average 450 kg Extensive grazing and mineralised mix	Oestrogen COCs recovered (n) Viable COCs (n) Embryos (n)	No significant difference between treatment and control groups in: COC recovery Viable COCs Significantly greater in treatment than controls: Embryos (5.9 ± 0.5 vs. 4.5 ± 0.4)	No sample size calculations Animal ethics approval is not stated No section to declare conflicts of interest Jadad score 2/5
23	Garcia et al., 2020	Brazil 2 × 2 crossover design 16 Bos indicus cows (Nelore) 3–10 years of age Not pregnant Not lactating Normal oestrus cycles 450 kg ± 25 kg Extensive grazing and supplement minerals	Oestrogen COCs recovered (n) Viable COCs (n) Embryos (n)	No significant difference between treatment and control groups in: COC recovery Viable COCs Significantly greater in treatment than controls: Mean embryos (6.5 ± 0.6 vs 4.8 ± 0.5)	No sample size calculations No randomisation Experiment 1 was excluded as the research question was not relevant to this study Only study to use higher dose luteolytic agent (530 μg of d‐clonprostenol) No information on washout period length Jadad score 1/5
24	Hidaka et al., 2018	Japan Experiment 2 Two‐period, two‐sequence crossover study Experiment 3 Paired difference one crossover study 15 Bos taurus cows (Wagyu) All animals fed silage All animals housed in stalls	Oestrogen Experiment 2 COCs recovered (n) Viable COCs (n) Experiment 3 COCs recovered (n) Embryos (n)	Experiment 2 Significantly greater in treatment than controls: Mean COCs recovered (28.9 ± 2.9 vs 21.8 ± 1.4) No significant difference between treatment and control groups in: Viable COCs Experiment 3 Significantly greater in treatment than controls: Mean COCs recovered (36.9 ± 5.5 vs 28.1 ± 3.7) Embryos (92 vs 28)	No sample size calculations No recruitment information No animal weight or BCS provided No information about OPU technician Only study to use E2 alone (no P4 or luteolytic agent) Used 1 mg E2 instead of 2 mg without stating animal weight The authors had the benefit of running Experiment 1 (not included) to deduce the optimal time to aspirate follicles to achieve the greatest number of medium‐sized follicles Jadad score 1/5
25	Kajaysri & Intrarapuk, 2024	Thailand Randomised controlled trial 30 Bos taurus cows (wagyu) Primiparous and pluriparous 250‐450 kg Temperature‐controlled barn Diet: Grass silage and 14% crude protein concentrate	Oestrogen COCs recovered (n) Viable COCs (n)	Significantly greater in treatment than controls: Number of medium‐sized COCs (group 4 and 5) Grade A and B COCs (group 4) Grade A COCs (group 5)	No sample size calculations Group 3 was excluded due to the use of superstimulatory hormones Did not investigate embryo production Jadad score 2/5
26	Ongaratto et al., 2015	Argentina 2 × 2 crossover design 45 Bos taurus (Angus and Brangus) Nonlactating Multiparous BCS between 2.5 and 3.5/5	Oestrogen COCs recovered (n) Viable COCs (n)	Experiment 1 Significantly greater in treatment than controls: Mean COCs recovered (7.9 ± 1.3 vs 3.2 ± 0.5) Mean viable COCs (5.2 ± 0.9 vs 2.1 ± 0.4)	No sample size calculations Inclusion and exclusion criteria and recruitment of participants is not stated No information on diet or participant housing Animal ethics approval not stated No information about OPU technician Experiment 2, 3 and 4 were excluded due to use of superstimulatory hormones No information on length of washout period between crossovers Did not investigate embryo production No section to declare conflicts of interest Jadad score 2/5
27	Ramos et al., 2010	Brazil Latin square design (3 × 3) Managed over 3 consecutive 28‐day studies Experiment 1 12 Bos taurus‐indicus crossbred heifers (Simental/Nelore) Pasture‐based system Mineral supplements Corn silage (15 kg/day)	Oestrogen COCs recovered (n) Viable COCs (n) Embryos (n)	Experiment 1 No statistical differences were found between 1x and 1x‐E2 for all parameters tested	No sample size calculations Animal ethics approval not stated OPU performed every 7 days No animal weight or BCS provided OPU was performed at 7‐day intervals Intravaginal progesterone device dose not stated Experiment 2 was excluded as the research question was not relevant to this study Hormones were provided for this study by a pharmaceutical company Jadad score 2/5
28	Batista et al., 2016	Brazil Randomised controlled trial 20 Bos indicus cows (Nelore) 485 kg ± 18 kg 47.4 ± 4.1 months BCS between 5.8 ± 0.31/9 All cows received the same total mixed ration diet	Gonadotropin‐releasing hormone COCs recovered (n) Viable COCs (n) Embryos (n)	Significantly greater in treatment than controls: COCs recovered (21.0 ± 2.3 vs 15.6 ± 1.9) Viable COCs (16.3 ± 2.1 vs 11.5 ± 1.6) No significant difference between treatment and control groups in: Embryos	No sample size calculations Only study that investigated the long‐term use of an intervention (180 days) Authors do not directly report on seasonal influence, except visually through graphs Denuded COCs were included as viable for IVEP Hormones were provided for this study by a pharmaceutical company Jadad score 2/5
29	Nawaz et al., 2022	Pakistan Randomised controlled trial 20 Bos indicus cows (Sahiwal) Nonpregnant Nonlactating Normal reproductive cycle BCS of 2.75–3.5 4–10 years of age	Progesterone COCs recovered (n) Viable COCs (n) Embryos (n)	Significantly greater in treatment than controls: COCs recovered per OPU session (5.31 ± 0.19 vs 3.58 ± 0.21) Viable COCs (4.54 ± 0.19 vs 3.06 ± 0.20) No significant difference between treatment and control groups in: Embryos	No sample size calculations No information on feed concentrate BCS scale of 5 is assumed The reader assumes that the OPU technician changed for last two OPU sessions Jadad score 1/5

Table 5.

The summary results of the three investigated clinical questions

Intervention	Studies (n)	Yes	Studies (n)	No
Question 1: Does synchronisation of follicular wave emergence increase the number of COCs recovered at OPU?
Oestrogen	2	Kajaysri and Intrarapuk (2024), Ongaratto et al. (2015)	4	Anchordoquy et al. (2024), Cavalieri et al. (2018), Garcia et al. (2020), Ramos et al. (2010)
Gonadotropin‐releasing hormone	1	Batista et al. (2016)	0
Progesterone	1	Nawaz et al. (2022)	0
Question 2: Does synchronisation of follicular wave emergence increase the COC quality recovered at OPU?
Oestrogen	3	Hidaka et al. (2018), Kajaysri and Intrarapuk (2024), Ongaratto et al. (2015)	4	Anchordoquy et al. (2024), Cavalieri et al. (2018), Garcia et al. (2020), Ramos et al. (2010)
Gonadotropin‐releasing hormone	1	Batista et al. (2016)	0
Progesterone	0	Nawaz et al. (2022)	0
Question 3: Does synchronisation of follicular wave emergence increase the production of embryos in IVEP?
Oestrogen	3	Cavalieri et al. (2018), Garcia et al. (2020), Hidaka et al. (2018)	2	Anchordoquy et al. (2024), Ramos et al. (2010)
Gonadotropin‐releasing hormone	0		1	Batista et al., 2016
Progesterone	0		1	Nawaz et al., 2022

Critical appraisal of the evidence

Following the appraisal, it was found that there are multiple strategies to synchronise cattle undergoing IVEP, with most of the evidence using oestrogen (E2) based protocols. Only two suitable studies were found that investigated the use of nonoestrogen‐based protocols, namely gonadotropin‐releasing hormone (GnRH) and progesterone (P4), with the scarcity of literature on these topics making it difficult to draw robust conclusions. The Jadad grading scale was used to assess the methodological quality of the included studies by investigating the randomisation procedures, animal removal and researcher blinding. ³⁰ Six papers scored 2/5 and the remaining three scored 1/5. This suggests that, according to the grading scale, the studies included could be perceived as of insufficient quality due to a lack of blinding of researchers, absence of explanation of the randomisation procedure and a lack of animal removal information. All studies, except for one, ²³ employed randomisation for group allocation.

The studies in this critically appraised topic (CAT) included a small number of animals, ranging from 12 to 45 individuals. All studies failed to disclose formal power and sample size calculations. There was a large discrepancy in the study durations and exposure to interventions. For example, one study performed synchronisation followed by OPU on day six with no further repeats, ²⁶ compared to a separate study that performed OPU once every 30 days over 180 days. ²⁸ The reproductive performance of animals in prolonged trial durations may be influenced by environmental confounders such as temperature change and seasonality/photoperiodicity. ³¹ Similarly, the prolonged exposure to exogenous hormones and repeated OPUs may affect normal physiology and the generalisability of the results.

The comparison between studies is made challenging by the variation in the hormone protocols and the dosages of drugs used. For example, varying doses of E2, formulations of prostaglandin F2α (i.e., cloprostenol vs dinoprost), and P4 are used throughout at differing time intervals. In addition, two studies failed to state the body condition status or liveweight, ²⁴ , ²⁷ and two studies failed to list the dietary intake of the animals involved, ²⁶ , ²⁹ both of which are of particular importance to successful bovine reproduction. ³² , ³³

Previous research has found that OPU intervals less than 14 days may risk carry over effects, reducing the number of viable and high‐quality oocytes available. ²¹ Six of the papers included in the appraisal are crossover designs, ²¹ , ²² , ²³ , ²⁴ , ²⁶ , ²⁷ with two studies having OPU intervals less than 14 days, ²¹ , ²⁷ and one study did not specify the crossover washout length. ²³ The time to new wave emergence following oocyte recovery (24–36 h), ³⁴ , ³⁵ combined with an 8–10 day duration of a follicular wave in most mature Bos taurus females, ³⁶ explains why OPU intervals or washout periods shorter than 14 days could influence the subsequent intervention/s.

Three studies did not specify whether the OPU technician(s) were consistent throughout the study. ²⁴ , ²⁶ , ²⁹ The level of technician competency can substantially affect the outcomes and may be a confounder when comparing results within and between studies. ¹³ Manipulation of ovaries with an active corpus luteum (CL) during OPU is more likely to induce haemorrhage and clots during aspiration. ³⁷ This may explain why recovery rates of cumulus‐oocyte complexes (COCs) retrieved from the ovary with a CL present can be reduced, ³⁸ whereas COCs retrieved from the ovary without a CL may exhibit higher recoveries. ³⁹ Thus, it is important that authors delineate which ovary (left or right) the oocytes are aspirated from if a CL is present and maintain consistency with OPU technicians for the reliability of results. Experimental factors beyond the scope of this current review are the OPU technique (e.g., transducer quality, vacuum pressure, needle diameter) and the protocol for IVM; however, we acknowledge that varying approaches were used and may be otherwise extraneous.

Most studies reported on the use of a systematic oocyte grading scale based on morphological assessment of oocyte ooplasm and number of cumulus layers, such as that reported by Seneda et al, and Barfield and Demetrio. ⁴⁰ , ⁴¹ This scale was used as the standard for classifying results, and where discrepancies in grading were found, they are reported in Table 4. For example, two studies accepted “denuded” oocytes (lacking cumulus cell layers at recovery) for IVM. ²¹ , ²⁸ The number of cumulus layers is positively correlated with embryo cleavage following fertilisation; therefore, inclusion of denuded oocytes could have jeopardised the success of embryo production in these studies. ⁴² The remaining studies utilised “good” (three or more cumulus layers) and “acceptable” (between one and two cumulus layers) oocytes for in vitro processing. Ultimately, this highlights how different IVEP systems adopt various protocols for oocyte selection and processing and therefore could confound results.

Two studies have included a combination of species or species‐crossbred animals in their study. ²⁶ , ²⁷ There are recognised physiological differences between Bos taurus and Bos indicus species that may affect their response to both hormonal synchronisation and suitability to IVEP. Namely, Bos indicus have a considerably higher antral follicle count (AFC), ⁴³ , ⁴⁴ and varying systemic concentrations of metabolic hormones (e.g., insulin and insulin‐like growth factor‐1) that influence oocyte and embryo quality. ³² Moreover, some Bos taurus species produce less endogenous E2 and have an increased clearance rate, resulting in lower circulating E2 than Bos indicus. ³² , ⁴⁵ Due to the grading scale and multi‐PICO framework, species‐specific stratification of results was not performed. Therefore, the lack of species‐specific analysis represents an important limitation, as interspecies differences may meaningfully affect the translation of these findings into clinical practice.

Discussion

Nearly a decade ago, the production of in vitro produced embryos surpassed those produced via conventional (in vivo) embryo transfer. For the first time, the IETS reported the production of more than 2 million embryos worldwide, with a significant increase in IVP embryos in Australasia from previous years. ¹ In line with this trend, the number of OPUs reported by registered practitioners is now more than 10 times that of conventional embryo flushes. ¹ Despite its global adoption, arguably IVEP remains relatively inefficient, with previous reports estimating that fewer than 20% of aspirated follicles ultimately yield an oocyte that develops into a high‐quality blastocyst. ⁴⁶ Superstimulation with exogenous gonadotropins such as FSH is used as a strategy to attempt to improve oocyte quality, with most studies reporting improved developmental competence of harvested oocytes through superstimulation. ¹⁷ , ¹⁸ The adoption of superstimulated OPU cycles varies geographically, with a higher proportion in North America and Europe, compared to other regions such as South America, where nonstimulated cycles predominante. ¹ This is probably a reflection of animal demographics, as Bos indicus cattle innately have a significantly higher AFC, housing a larger existing oocyte population likely to have acceptable quality oocytes. ⁴⁴ At times, porcine pituitary‐derived FSH has been limited in market supply, may carry negative societal perceptions and significantly increases costs associated with IVEP. Also, currently, there are no commercially available recombinant FSH products on the market for cattle, further emphasising the reliance and dependency on those animal‐derived. ⁴⁷ Therefore, it is warranted to investigate alternative strategies that can improve outcomes in IVEP programmes.

The bovine oestrus cycle consists of wave‐like emergences of follicles every 8–10 days, with some variation noted according to age and between species. ³⁶ By the fourth day of the follicular wave, subordinate follicle growth plateaus, due to the nadir of FSH, ³⁶ with many medium‐sized follicles (6–10 mm) having developed. This point in the oestrus cycle offers a homogeneous pool of growth phase follicles housing oocytes that are likely to possess meiotic competence. ⁴⁸ , ⁴⁹ , ⁵⁰ It has been shown that oocytes from subordinate/regressing follicles in the presence of a dominant follicle exhibit lower embryo development rates than those from follicles in the active growing phase in most bovine IVEP systems. ¹² Adopting strategies, such as inducing FWE by implementing oestrous synchronisation protocols and timing OPU to harvest a population of oocytes from follicles at an optimum stage of development, could therefore be a physiologically sensible alternative to superstimulated cycles.

Strategies to implement FWE include both mechanical and hormonal protocols. A mechanical process termed Dominant Follicle Reduction or Ablation (DFR/DFA) via transvaginal ultrasound guided aspiration of visible follicles greater than 5 mm has been shown to be a highly reliable means of resetting a new follicular wave in 24–36 h. ³⁴ , ³⁵ A controlled trial investigated DFR efficacy prior to OPU, ⁵¹ which found that OPU performed 72 h after DFR in non‐superstimulated animals could significantly increase embryo production compared to controls; however, this technique is not without its limitations. For instance, the technical difficulty and the requirement of a veterinarian/technician may limit its practicality in large‐scale or field operations. Additionally, there is the rare risk that residual follicles may be hormonally active and negatively impact the timing of FWE. ⁵² Although a promising strategy, it unfortunately did not meet the specific inclusion criteria under the Jadad scale as it was a nonrandomised clinical trial.

Gonadotropin‐releasing hormone (GnRH) can synchronise FWE by inducing ovulation and/or luteinisation, with FWE occurring 24–48 h following administration. ⁵³ One study investigated this premise in lactating Bos taurus dairy cows, finding that GnRH synchronisation significantly improved oocyte number and quality at OPU and embryo production, ⁵⁴ although it again did not meet the inclusion criteria, being a nonrandomised controlled trial. A reported limitation of this technique is that only 70% of lactating dairy cows will ovulate in response to a single GnRH injection. ⁵⁵ This is attributable to the size and developmental stage of the existing antral follicles at the time of GnRH administration. For instance, follicles that are either too small (i.e., greater than ~10 mm for Bos taurus) or previously dominant atretic follicles in the regression phase that are also refractory to ovulation induction. ⁵³ , ⁵⁶ , ⁵⁷ An alternative application of GnRH is the administration of a long‐acting subcutaneous implant to downregulate the hypothalamic‐pituitary‐gonadal (HPG) axis, ²⁸ and the subsequent desensitisation of the anterior pituitary, resulting in the absence of FSH, an LH surge and ovulation. ⁵⁸ One included study investigated this over 180 days, resulting in an increased number of small COCs and the quality of oocytes in Bos indicus cows; however, it did not improve embryo production compared to controls. ²⁸ Previous research in Bos indicus Nelore cows has shown that 20% of oocytes collected from small follicles (1–3 mm) do possess developmental competency, compared to 35% of medium follicles, and 63% for large follicles. ⁵⁹ The findings suggest that in Bos indicus cows, the use of long‐term GnRH agonists to downregulate the HPG axis, resulting in the production of many small follicles, may not be an economically viable option for IVEP systems and could interfere with fertility.

The coadministration of exogenous E2 and P4 can induce atresia of the dominant follicle within 36 h and result in the emergence of a new follicular wave in 3–5 days. ⁶⁰ Unlike many other countries, thankfully, Australian veterinarians remain able to prescribe exogenous E2 for reproductive purposes in certain instances (i.e., non‐European Union Accredited herds and nonlactating dairy cattle). ⁶¹ Although limited literature was available for alternative techniques such as DFR and GnRH‐based protocols, a comparatively larger body of evidence exists for E2‐based protocols. Overall, the cumulative findings of this CAT suggest that oestrogen‐based synchronisation prior to OPU provides no clear irrefutable improvements for the three metrics investigated (COC recovery, COC grade and embryo production in vitro). Although only two of the seven studies uniformly concluded that none of the three aforementioned outcome measures were improved, findings from the remaining five E2 + P4 studies were either less definitive or incomplete. For instance, two studies reported no improvement in oocyte number or morphological grade following E2 + P4 synchronisation, although both found increased embryo production, ²² , ²³ which may indicate a modulatory effect of E2 on developmental competence independent of oocyte yield or morphology. Conversely, two studies did in fact report enhanced oocyte yield and quality following E2 + P4 synchronisation, although neither extended their analysis to the embryo production stage, limiting interpretation of developmental outcomes. ²⁵ , ²⁶ Had these studies investigated embryo production, the results of this study may have yielded a more pertinent overall conclusion.

A species‐specific response is also possible, with Bos taurus donors showing improvements in oocyte number and grade following E2 + P4 synchronisation, while Bos indicus donors appear to derive greater benefit at the level of embryo production. The aforementioned interspecies differences in reproductive physiology may account for the greater responsiveness of Bos taurus to E2‐mediated alterations in oocyte parameters; however, the observed enhancement in embryo production in Bos indicus remains mechanistically unclear and warrants further investigation. Variability in study populations and methodological design complicates cross‐study comparison and interpretation. Accordingly, future investigations should incorporate species‐specific methodologies that account for interspecies reproductive physiological differences, a limitation inherent to the present appraisal. The lack of consistency among results of oestrogen‐based protocols invites speculation about the methodology and/or species differences of the included studies and suggests future work should pursue information about embryo production where possible.

Despite the wide adoption of IVEP, there is no standardised approach to the IVM process of bovine embryos, despite most base media being somewhat consistent. It is documented that progesterone (P4), amongst other potential media components such as synthetic oviductal fluid and gonadotrophin, ⁶² is important in intracellular signalling and IVM processing of bovine embryos. ⁶³ This suggests that prior in vivo exposure to P4 may influence the success of an IVEP protocol. One study investigated independent P4 use on its own directly, ²⁹ finding that in Bos indicus cattle, oocyte quality was improved by the presence of a controlled internal drug release device containing 1.9 g of P4 during OPU, although there was no effect on embryo production. Although P4 exposure varied among E2 + P4 combination protocols in terms of dose, duration and timing, the collective findings suggest that progesterone (irrespective of endogenous or exogenous origin) is unlikely to have acted as a major confounder of the observed results.

Despite most studies following the conventional morphological assessment of recovered oocytes after OPU for determination of quality, ⁴¹ only two studies followed through to confirmation of pregnancy. ²² , ²³ The simplest definition of developmental competencee would be the ability of an oocyte to be successfully fertilised and develop into a quality blastocyst, ⁶⁴ however, a more complete and biologically relevant endpoint would include all the way to a live birth. ¹⁰ Given the assessment criteria applied in this appraisal, a definitive conclusion regarding the effect of synchronisation on true developmental competence cannot be established, as live calf birth rate was not included as a predefined outcome measure. Nonetheless, embryo development and pregnancy diagnosis remain industry‐relevant endpoints. Future high‐quality research is necessary to investigate the efficacy of oestrogen‐based and nonoestrogen‐based synchronisation protocols prior to OPU. Where feasible, studies should follow through to pregnancy diagnosis and parturition, be species‐specific, have an adequate washout period (>14 days), maintain consistent OPU technicians and apply clear blinding and randomisation procedures to improve the quality of evidence.

Clinical bottom line

Findings from critical appraisal suggest that there is equivocal evidence for the use of oestrogen‐based synchronisation protocols in nonsuperstimulated cattle undergoing OPU‐IVEP. The studies included have limitations in study design and population size, and the neutrality of results provides an overall low certainty for the irrefutable recommendation for this strategy as an alternative to superstimulation in OPU‐IVEP systems. Although other methods of FWE were investigated, they were excluded from the final summary due to incompatible study design and suitability for the Jadad grading scale. Further research is needed to investigate nonoestrogen alternatives, as currently there is insufficient evidence that shows a clear, tangible benefit to employing synchronisation of follicular wave emergence to improve outcomes in all IVEP systems.

Conflicts of interest and sources of funding

The authors declare no conflicts of interest or sources of funding for the work presented here.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.