Utilizing the Laparoscopic Ovum Pick-Up Method for InVitro Embryo Production in Prepubertal Anatolian Native Black Calves: A Promising Approach for Gene Banks and Endangered Species.

Comparison of ultrasound-guided vs laparoscopic transvaginal ovum pick-up (OPU) in simmental heifers

Prolonging the interval from ovarian hyperstimulation to laparoscopic ovum pick-up improves oocyte yield, quality, and developmental competence in goats

The aim of the present study was to evaluate the correlations between biochemical serum profile with total number of aspirated follicles (AF), oocytes recovered (OR), number of corpus lutea (CL), oocyte morphology quality, and oocyte in vitro maturation (IVM) rate. Eighteen goats were randomly divided into 3 groups based on treatment diets, with different fat sources [soy oil (SG, n = 6), linseed (LG, n = 6), and Megalac® (MG, n = 6)], formulated with 40% concentrate, 60% corn silage, of which 4% was ether extract from fat sources. The animals were submitted to a 15-day adaptation period and then a 70-day experimental period. The laparoscopic ovum pickup (LOPU) was performed after 36 h of an ovarian superstimulation protocol [FSH (80 mg) + eCG (300 IU)] on Day 42 (LOPU1) and Day 70 (LOPU2) of the trial. The number of AF, and the presence and number of CL were evaluated during the LOPU procedure. Blood samples were taken at 14 days, 7 days, and just before each LOPU, and grouped as P1 (mean of blood samples results obtained before LOPU1) and P2 (LOPU2); serum metabolites analysed were total cholesterol, triglycerides, aspartate aminotransferase (AST), creatine kinase, and plasma glucose. The recovered oocytes were classified by morphological appearance, and only G1 (homogenous ooplasma and at least 3 layers of cumulus cells), G2 (homogenous ooplasma and at least 1 complete layers of cumulus cells), and G3 (homogenous ooplasma and incomplete layers or no cumulus cells) were submitted to IVM. The oocytes were incubated in maturation media (TCM 199 supplemented with 10% heat inactivated goat serum) at 38.5°C and 5% CO2 for 27 h. Then, oocytes were subsequently fixed and stained on Hoechst 33342 and analysed using a fluorescent microscope for nuclear maturation. The IVM and analysis were performed for each individual. Pearson correlation test (P < 0.05) was applied in R® software between serum metabolites and LOPU variables. The triglycerides, creatine kinase, and glucose were not correlated with any LOPU variables. The total cholesterol was negatively correlated with G1, G2, and CL, and positively correlated with OR and G3 with determination coefficients (R) of –0.28, –0.38, –0.24, 0.25, and 0.44, respectively. The AST were negatively correlated with AF, G1, G2, and IVM, with R of –0.11, –0.43, –0.13, and –0.16, respectively. In conclusion, these results are suggestive that high serum levels of cholesterol and AST may interfere with oocyte quality, indicating a negative hyperlipidemia effect on reproductive efficiency as reported in cows.Financial support was provided by CNPq and FAPESP.

A strategize step in oocyte recovery is important and beneficial in producing successful production of high-quality Cumulus Oocyte-Complexes (COC) for in vitro maturation, fertilization and culture (IVMFC), nuclear transfer and other emerging technologies. Experiments were conducted to compare Oocyte Recovery (OR) between Laparoscopic Ovum Pick-Up (LOPU) and ovarian slicing and to evaluate the effects of OR cycle, hormone stimulation, oestrus synchronization, breeds, liveweight and age of goats on the quantity and quality of oocytes obtained. In Experiment 1, oocytes were recovered from abattoir-derived ovaries by the slicing method. The ovaries were transported to the laboratory for immediate harvesting. The slicing method yielded significantly (p<0.05) larger number of oocytes per ovary than LOPU (22.53±2.78 vs. 6.10±0.46). However, the proportion of Grade A oocytes was highest from the LOPU technique (30.51±4.14) compared to the slicing method (19.07±3.57). LOPU resulted in good quality Cumulus Oocytes Complexes (COC) with more than 5 layers of cumulus cells. Experiment 2 consisted of three OR cycles (OR-1, OR-2 or OR-3); four durations of oestrus synchronization using CIDR (Day-10, -14, -17 or -21); three goat breeds (Boer Crossbred, Mixed breed or Katjang); 4 age groups (Young, Mature, Old or Very Old) and 4 levels of body weight (20, 21-29, 30-37 or 38 kg). Goats were synchronized using Control Internal Drug Release (CIDR) combined with 125 jig Estrumate and hyperstimulated with 70 mg FSH and 400 IU hCG after the removal of CIDR 24 h later. There was no significant effect of OR cycle, CIDR removal goat breed, age or body weight on the total number of oocytes recovered per ovary. Katjang goats yielded a higher proportion of Grade A oocytes compared to Boer Crossbreds and mixed breeds which had a higher proportion of Grade C oocytes (p<0.05). Age and weight of animals had no significant effect on the quality of oocytes recovered. Grade A and B oocytes were obtained in OR-1, whilst the repetition of LOPU resulted in an increment of Grade C oocytes in OR-2 and OR-3, respectively. Day-10 and -21 gave the highest cumulative percentage of 58 and 64% for Grade A and B oocytes, respectively. Under the conditions of the experiment, LOPU yielded better quality oocytes from the Katj ang goats using less or no OR repetition, with oestrus synchronization at 10 or 21-Day. © 2013 Academic Journals Inc.

Laparoscopic ovum pickup (LOPU) is a modern technique that may be used in programs of sheep and goat in vitro embryo production. The effects of repeated recoveries and follicular diameter on the efficiency of LOPU were evaluated in these species. In six Merino donor sheep and five Criolla goats, ovarian stimulation for follicular development was performed by administering a single dose of 60 mg NIH-FSH-P1 (Folltropin®; Bioniche, Canada) and 300 UI of eCG (Novoromon 5000®; Syntex, Argentina). Every four days, a total of 4 LOPU in sheep and 3 in goats were performed 24 h after each ovarian stimulation treatment. The intravaginal sponge with 60 mg of medroxyprogesterone (Esponjas Vaginales Syntex®; Syntex, Argentina) were removed after the last LOPU. Follicular fluid was aspirated under laparoscopic observation according to the follicle diameter with a cannula for intrauterine artificial insemination without a vacuum pump. The number and size of follicles, oocyte quantity and quality and recovery rate were evaluated. A total of 566 follicles were aspirated (14.4 ± 2.4 follicles/sheep and 14.6 ± 7.5 follicles/goat), yielding a total of 263 oocytes (6.8 ± 1.5 oocytes/sheep and 6.6 ± 2.9 oocytes/goat) and a recovery rate of 47% in sheep and 46% in goats (Table 1). In both species, there were no significant differences in the number of aspirated follicles, recovered oocytes, overall recovery rate, and the recovery rate of good quality oocytes in the repeated LOPU. The oocyte recovery rates in sheep differed significantly according to the follicular size (55% in large follicles, 36% in small follicles; P &lt; 0.05), whereas in goats recovery rates of oocytes related to follicular size showed no significant differences (42 and 54% for large and small follicles, respectively). Follicular sizes from which oocytes were obtained was not a determining factor in the recovery rate of good quality oocytes. The technique used would allow effective and repeated oocyte recoveries for in vitro embryo production in sheep and goats. Table 1. Repeated recoveries by laparoscopic ovum pickup (LOPU) in sheep and goats

Whether we are collecting oocytes from valuable donors as part of a program of in vitro embryo production (IVP) and/or to promote advances in emerging biotechnologies as cloning and transgenesis, the success rate depends on the development of methodological aspects of recovering oocytes. To succeed, sufficient number of good quality oocytes is the prerequisite for various reproductive techniques and laparoscopic ovum pick up (LOPU) is the recommended technique for obtaining them from live goats. However, the variability of the quantity and quality of the oocytes collected still limits the large-scale use of this technology. Under the current conditions, too large variability is reported with oocyte recovery rates ranging from 40 to 90%, and the number of harvested oocytes per female between 4 and 14, in different laboratories. This variability can depend on either intrinsic characteristic of the donors, such as breed, age or on aspects that we might be able to control, such as stimulation treatment, type of needle, aspiration pressure, among others. We believe that new investigations should contribute to significant improvement of LOPU yield. This review aims to report different factors influencing goat donor response for LOPU, presenting main steps for oocytes recovery as well as technical modifications proposed for improving LOPU efficiency. Furthermore, it is aimed to discuss about the potential use of goat oocytes after their recovery by LOPU and present overall results in goat IVP worldwide.

The present study was conducted to evaluate the follicular response and oocyte yield following repeated gonadotrophin stimulation and laparoscopic aspiration in goats and to assess the effects of the time interval between procedures and season. A total of 98 adult goats were subjected to laparoscopic ovum pick-up (LOPU) five consecutive times in a transgenic production programme. Oestrus was synchronised by means of intravaginal sponges inserted for 10 days coupled with 125 microg cloprostenol 36 h before sponge removal and LOPU, and follicular development was stimulated with 80 mg follicle stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. No difference was detected in the response for LOPUs 1, 2, 3 and 4. Although a small decrease in response was detected at LOPU 5 (P < 0.05), the numbers of follicles aspirated and oocytes recovered were not different from those at LOPU 1 and LOPUs 1 and 4, respectively. With respect to time interval between LOPU and season, all intervals and seasons produced acceptable responses, with no difference in follicles aspirated and oocytes recovered between intervals and seasons. These results indicate that LOPU may be repeated up to five times in goats at different intervals and in different seasons with little or no important change in overall response.

Simple summaryIn this study, minimally invasive laparoscopic methods of recovering oocytes in goat and sheep (L-OPU, laparoscopic ovum pick-up) were developed and improved. Applying the laparoscopic technique allows animal welfare to be significantly improved while still maintaining high effectiveness of the method. The method allows a high number of good quality oocytes to be obtained and additionally reduces the invasiveness of the method and tissue damage which occurs during the operation to a minimum. It requires a short time and allows animals to return to the herd immediately after the operation has been completed. Additionally, it also gives the opportunity for maximum use of the animal’s genetic capability. The limitation of using the L-OPU method in goats is its variable and changing effectiveness. That is why it is crucial to develop an effective and repeatable method. The study compared several variants of the method including varying techniques of conducting the operation, various tools for recovering oocytes, and different plans of hormonal stimulation. As a result, an optimal method of recovering oocytes was developed. The research may be of great importance in improving the welfare of animals and increasing the effectiveness of biotechnological methods used in goat and sheep breeding as well as in the area of research.The laparoscopic method of recovering oocytes in goats and sheep is one of the minimally invasive methods used in the biotechnology of animal reproduction. It allows for good quality oocytes that are suitable for in vitro maturation and fertilization to be recovered. The limitation of using the laparoscopic ovum pick-up (L-OPU) method in goat and sheep is its changing effectiveness and the lack of repeatability of results, as well as the varying effectiveness of different variants of the method. Therefore, it is necessary to develop effective non-invasive techniques allowing for multiple good quality oocyte recovery that would be suitable for in vitro maturation and fertilization. In this study, four different L-OPU variants were described in goats and sheep. Various techniques of recovering oocytes were discussed, including the techniques of conducting the operation, various tools for recovering oocytes, and different plans of hormonal stimulation. Recovery rates were 35% (Variant I), 57% (Variant II), 72% (Variant III), and 67% (Variant IV). After evaluation, 94% (both Variant I and II), 93% (Variant III), and 84% (Variant IV) of the oocytes were qualified for in vitro maturation. The results of the study show that the proposed technique of laparoscopic recovery of oocytes allows a sufficient number of ovarian cells suitable for in vitro culture to be obtained and as a consequence it makes them useful in in vitro maturation/in vitro fertilization (IVM/IVF) programs or cloning. The method allows for a fast and effective conduct of the operation in a living donor with minimal invasiveness while preserving the excellent condition of animals.

Oocytes are the main raw materials for in vitro embryo production (IVP) experiments. Therefore, the success of any IVP program in goat production, either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) largely depends on the continuous supply of quality oocytes in optimum quantity. A number of methods are currently used for oocyte recovery (OR) from live or slaughtered goats. Although, abattoir is the most easy and cheapest source of oocytes, however, in certain countries especially in Malaysia, abattoir source is extremely limited due to low slaughtering activities as a consequence of shortage of breeding stock. In Malaysia normally older goats or goats those are culled for breeding are slaughtered for meat. Therefore, the quality of oocytes recovered from ovaries of these goats is generally lower. On the contrary, OR from live goats using laparoscopic ovum pick-up (LOPU) technique provides many advantages, for example, OR can be repeated 3-5 times in the same goat at interval as short as a week or less, OR can be done in prepubertal and aged goats, if prepubertal goat is used for OR then generation intervals will be reduced, LOPU coupled with IVP can become an effrcient method of early propagation of valuable goats and LOPU can overcome limitations frequently associated with multiple ovulation-embryo transfer (MOET). Therefore, LOPU can be an alternative and efficient OR method in goat not only for Malaysia but also for other countries. The current study will discuss oocyte recovery methods with special reference to LOPU and grading of recovered oocytes in goats. © 2008 Asian Network for Scientific Information.

Simple SummaryThis study investigates the use of Chongming goats for optimizing laparoscopic ovum pick-up (LOPU) and in vitro embryo production (IVEP). Key metrics such as the recovery rate of cumulus–oocyte complexes (COCs), the number of ovarian follicles, and the quality of collected COCs are assessed to refine estrus synchronization and superovulation techniques. Additionally, the study examines the effectiveness of LOPU using indicators like cleavage and blastocyst rates to enhance IVEP efficiency, aiming to develop a robust LOPU technology system for small ruminants.The application of laparoscopic ovum pick-up (LOPU) and in vitro production of embryos (IVEP) technologies has opened up a new path for purebred breeding and breed improvement in goats. However, due to the complexity of the procedures and multiple influencing factors, these technologies have not been widely adopted in goat production. This study explores factors affecting the efficiency of LOPU in goats by comparing the use of controlled internal drug release (CIDR) for estrus synchronization, conventional FSH versus long-acting recombinant ovine FSH (R-FSH) for superovulation, and the timing of LOPU at 48 h, 60 h, and 72 h of follicular development. The metrics evaluated included the recovery rate of cumulus–oocyte complexes (COCs), the average number of ovarian follicles, the average number of COCs, and the average number of available COCs. The results demonstrated that the efficiency of LOPU was significantly higher with two doses of R-FSH compared to the conventional FSH superovulation protocol and the control group (p < 0.05), with two doses of R-FSH providing a higher LOPU efficiency than one dose (p < 0.05). Using CIDR for estrus synchronization showed no significant difference in LOPU efficiency compared to the non-CIDR group. Similarly, the efficiency of LOPU showed no significant difference between the hormone treatments at 48 h, 60 h, and 72 h. By exploring and optimizing the factors influencing LOPU, we ultimately established a LOPU technology system for goats that meets the production needs of small ruminants.

Simple SummaryOne of the main obstacles to the in vitro production of embryos in goats is the low ovarian response to hormonal treatments and low oocyte quality. Thus, several strategies have been performed to improve the reproductive performance of goats, including the development of new hormonal protocols as well as the use of other drugs that act directly or indirectly on reproductive function. In this experiment, we tested the use of a hormonal protocol aimed at maximizing the ovarian response and, in parallel, an angiotensin-converting enzyme (ACE) inhibitor was administered daily as an adjuvant. In recent years, the renin-angiotensin system has been shown to play an important role in reproductive function, especially in follicular development. We found that administration of the ACE inhibitor affected the ovarian response in multiparous goats, with more visible follicles, and had no effect on oocyte quality or during embryonic development, thus being a possible alternative to improve goat reproductive response.The aim of this work was to determine the effect of enalapril maleate administration, during oocyte recovery by serial laparoscopic ovum pick-up (LOPU), on the ovarian response and in vitro embryo production (IVP). Twenty cross-bred goats were allocated equally into two groups: Nulliparous and Multiparous. In each group, five animals were selected to receive daily doses of enalapril maleate during the hormonal protocol. Estrus was synchronized by a PGF2α analog, followed 48 h later by insertion of an intravaginal device with progesterone. Forty-eight hours after, a single dose of FSH/eCG was administered. The FSH/eCG doses were repeated three times, on every four day. Oocytes were recovered by LOPU 24 h after each FSH/eCG dose. Viable oocytes were matured in vitro, to be parthenogenetically activated and cultured for 72 h to the cleavage stage. The drug treatment increased the proportion of total follicles observed at LOPU (p < 0.01) in multiparous goats. In both parity groups, enalapril administration had no effect on the proportion or quality of oocytes recovered. Furthermore, the number of embryos cleaved was similar between the groups. Thus, enalapril maleate affected the ovarian response in multiparous animals only and had no effect on the oocyte quality or IVP.

This study was aimed at the development of FSH administration protocols using a slow-release formulation (SRF, Bioniche, Belleville, Ontario, Canada) for simplification of multiinjection protocols used for goat superovulation in multiple-ovulation embryo transfer (MOET) and laparoscopic ovum pickup (LOPU) programs. Moreover, FSH-SRF was also tested as a replacement for eCG in oestrus synchronization programs, as a potential solution for countries where eCG is not licensed and for animals that have become refractory to eCG after multiple treatments. In the MOET group, 20 goats were synchronized with CIDR (Pfizer, Montreal, Quebec, Canada) and superovulated, and embryos were collected by mid-ventral laparotomy 6 days after a single intrauterine insemination with fresh semen. Half of the donors (control) were treated with a standard superovulation regimen consisting of a total 160 mg of FSH (Folltropin, Bioniche) administered in 8 decreasing-dose injections at 12-h intervals, starting 48 h before CIDR removal. The other half (SRF group) received the same total amount of FSH in 2 injections administered 2/3 at –48 h and 1/3 at CIDR removal. In the LOPU group, 10 control goats were treated with a total 80 mg of FSH administered in 3 equal injections at 12-h intervals, starting –36 h from LOPU, whereas the SRF-FSH group received the total amount in a single injection at –36 h from LOPU. Finally, to evaluate the effectiveness of SRF-FSH in oestrus/ovulation synchronization, 20 goats were injected at CIDR removal with either 400 IU of eCG (Pregnecol, Bioniche) or 20 mg of FSH-SRF or 40 mg of FSH-SRF and evaluated by the presence of a morphologically sound corpus luteum (CL) by laparoscopy on day 6 from oestrus. No significant differences (ANOVA, P &gt; 0.05) were obtained between the control and SRF groups in terms of average number of CL (9.1 v. 10.0), ova recovered (6.2 v. 7.7), and transferable embryos (5.4 v. 6.5) in the MOET part of the study. Similarly, no differences were observed in the average number of follicles for aspiration (17.8 v. 17.9), oocytes recovered (13.7 v. 14.0), and recovery rate (76.9 v. 78.2%) in goats subject to LOPU. In the oestrus synchronization group, all animals were observed in standing heat in the period from 36 to 60 h post-CIDR removal. The average number of CL did not differ among the 3 treatment groups (1.9 v. 2.0 v. 2.4). One case of no ovulation (10%) and one of premature luteolysis (10%) were found in the control group, whereas 100% of the goats in the FSH-SRF groups had normal CL present. These results indicate that FSH-SRF can significantly reduce the amount of labour associated with multiinjection hormonal regimens (MOET, LOPU) without loss in donor responses and can be used for oestrus synchronization as an alternative to eCG-based protocols. All these results were produced during the Canadian winter; further studies will be conducted in other seasons of the year to explore the effect of goat reproductive seasonality in the response to the FSH-SRF treatment strategies described.

Repeated hormonal treatment and laparoscopic ovum pick-up followed by in vitro embryo production in goats raised in the tropics

Laparoscopic ovum-pick up and in vitro embryo production in gonadotropin-stimulated gilts: Preliminary results and envisioned applications

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up–derived oocytes have different kinetics and requirements regarding maturation media